Methods for Generating Analogs of Coenzyme A

ABSTRACT

Methods to generate analogs of coenzyme A in vivo are disclosed. The methods to generate analogs of coenzyme A in a cell comprise reacting pantetheine or a derivative thereof with a reporter to form labeled pantetheine or a derivative thereof, contacting the cell with the labeled pantetheine or derivative thereof such that the labeled pantetheine or derivative thereof enters the cell, phosphorylating the labeled pantetheine or derivative thereof to form phosphopantetheine or a derivative thereof, adenylating the labeled phosphopantetheine or derivative thereof to form a labeled dephosphoCoenzyme A or derivative thereof, and phosphorylating the 3′-hydroxyl of the labeled dephosphoCoenzyme A or derivative thereof to form a labeled coenzyme A analog or derivative thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 11/485,247, filed Jul. 11, 2006, which claimsbenefit to U.S. Provisional Application No. 60/698,333, filed Jul. 11,2005, which is incorporated herein by reference in its entirety.

STATEMENT OF U.S. GOVERNMENT SUPPORT

This invention was made by government support by Grant No. MCB0347681from the National Science Foundation and Grant No. RO1GM075797 from theNational Institutes of Health. The Government has certain rights in thisinvention.

FIELD OF THE INVENTION

This invention relates generally to the field of cell biology. Methodsto covalently label proteins in vitro and in vivo by way of labeledcoenzyme precursors are disclosed.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. These cited publications are incorporated by referenceherein, in its entirety and for all that they illustrate.

Selective chemical control of biochemical processes within a living cellenables the study and modification of natural biological systems in waysthat may not be obtained through in vitro experiments (Cook et al., C.R.Bioorg. Med. Chem. 10: 829, 2002; Chen et al., Curr Opin Biotechnol.16:35, 2005). Accordingly, access to promiscuous metabolic pathways hasprovided a unique chemical entry into small molecule engineering in vivo(Mahal et al., C.R. Science 276:1125, 1997). A method for covalentreporter labeling of carrier proteins using promiscuousphosphopantetheinyltransferase (PPTase) enzymes and reporter-labeledcoenzyme A (CoA) has recently been described (La Clair et al., M.D.Chem. Biol. 11:195, 2004). Until now, this method has been limited to invitro and cell-surface protein labeling, as CoA derivatives have notbeen shown to penetrate the cell (La Clair et al., M.D. Chem. Biol.11:195, 2004; Mercer et al., ChemBioChem 2005; George et al., Am. Chem.Soc., 126: 8896, 2004; Yin et al., Am Chem. Soc. 126: 13570, 2004; Yinet al., Am Chem. Soc. 126: 7754, 2004). To overcome this obstacle,labeled metabolic precursors may be delivered to the cell culture, whichresults in cellular uptake and metabolic conversion into active, labeledCoA derivatives. In the process, a chemoenzymatic route to proteinmodification via a four-step sequence is established.

The chemical synthesis and activity of CoA has been studied for wellover a century, yet the full biosynthesis of the cofactor has onlyrecently been elucidated in prokaryotes and eukaryotes (Mishra et al.,Chem. Rev., 100: 3283, 2000, Mishra et al., Bacteria, 183: 2774, 2001;Martin et al., Biochem. Biophys. Res. Commun. 192: 1155, 1993; Strausset al., Biol. Chem. 276:13513, 2001; Daugherty, et al., Biol. Chem.277:21431, 2002; Kupke et al., Biol. Chem. 278:38229, 2003; Zhyvoloup etal., Biol. Chem. 277:22107, 2002 and Worrall et al., Biochem. 215:153,1983). CoA is biosynthesized from vitamin B5 by five enzymes in E. coli,CoaA-CoaE, while eukaryotes contain a fusion of CoaD and CoaE,PPAT/DPCK. Knowledge of the substrate specificity of these enzymesremains incomplete, although some evidence points to promiscuity withinthis pathway. Early studies on CoaA indicated that the enzyme will alsoaccept pantetheine as a substrate (Abiko, Biochem 61:290, 1967 andShimizu et al., Biol. Chem. 37:2863, 1973). This ability has since beenused in the chemoenzymatic synthesis of CoA analogs (Rudik et al.,Biochemistry 39:92, 2000; Martin et al., J, Am. Chem. Soc., 116:4660,1994; Schwartz et al., Biochemistry 34:15459, 1995 and Nazi et al.,Anal. Biochem., 324:100, 2004). This permissiveness suggests the abilityof the CoA biosynthetic pathway to convert reporter-labeled pantetheineto reporter-labeled CoA in vivo. To this end, the synthesis offluorescently-labeled pantetheine analogs provides a direct link toreporter-labeled post-translational modifications (Mandel, A L et al.2004).

SUMMARY OF THE INVENTION

The present invention features methods to generate analogs of coenzyme Ain vitro and in a cell. In one aspect of the invention, coenzyme Aanalogs are generated by reacting pantetheine or a derivative thereofwith a reporter to form labeled pantetheine or a derivative thereof,phosphorylating the labeled pantetheine or derivative thereof to formphosphopantetheine or a derivative thereof, adenylating the labeledphosphopantetheine or derivative thereof to form a labeleddephosphoCoenzyme A or derivative thereof, and phosphorylating the3′-hydroxyl of the labeled dephosphoCoenzyme A or derivative thereof toform a labeled coenzyme A analog or derivative thereof.

In a detailed embodiment, the pantetheine or derivative thereofcomprises three modules, a ω-functionalized amine, sidechain, anα-aminoacid, or β-aminoacid, or linker, and a modulator. Theω-functionalized amine, sidechain, α-aminoacid β-aminoacid, linker, ormodulator may comprise a reporter. In a further aspect, pantetheine oran analog thereof, can be synthesized by microwave-assisted nucleophilicring opening of pantolactone, and may further comprise a reporter. Thereporter can be an affinity reporter, colored reporter, fluorescentreporter, magnetic reporter, radioisotopic reporter, peptide reporter,metal reporter, nucleic acid reporter, lipid reporter, glycosylationreporter, reactive reporter, enzyme inhibitor, biomolecular substrate,and the like, or a precursor to any of such reporters.

In a further detailed embodiment, the steps of the inventive method areenzyme-catalzyed. Phosphorylation of the labeled pantetheine orderivative thereof can be catalyzed by a kinase such as pantothenatekinase. Adenylation of the labeled phosphopantetheine or derivativethereof can be catalyzed by an adenylyltransferase such asphosphopantetheine adenylyltransferase. Phosphorylation of the3′-hydroxyl of the labeled dephosphoCoenzyme A or derivative thereof canbe catalyzed by dephospho-CoA kinase.

In a still further detailed embodiment, the method to generate analogsof coenzyme A further comprises reacting the labeled coenzyme A analogor derivative thereof with a carrier protein domain to form a labeledprotein. The carrier protein domain comprises a fusion construct betweena peptide or carrier protein domain and a protein of interest. Thisreaction can be catalyzed by an enzyme such as those in the class ofphosphotransferases. An example of such a phosphotransferase compatiblewith the methods of the present invention is4′-phosphopantetheinyltransferase.

Another aspect of the present invention features methods to generatecoenzyme A analogs in a cell. Such coenzyme A analogs are generated byreacting pantetheine or a derivative thereof with a reporter to formlabeled pantetheine or a derivative thereof, contacting the cell withthe labeled pantetheine or derivative thereof such that the labeledpantetheine or derivative thereof enters the cell, phosphorylating thelabeled pantetheine or derivative thereof to form phosphopantetheine ora derivative thereof, adenylating the labeled phosphopantetheine orderivative thereof to form a labeled dephosphoCoenzyme A or derivativethereof, and phosphorylating the 3′-hydroxyl of the labeleddephosphoCoenzyme A or derivative thereof to form a labeled coenzyme Aanalog or derivative thereof.

In a detailed embodiment, the cell is a eukaryote, prokaryote, orarchaebacterial cell.

In a further detailed embodiment, the pantetheine or derivative thereofcomprises three modules, a ω-functionalized amine, sidechain, anα-aminoacid, or β-aminoacid, or linker, and a modulator. Theω-functionalized amine, sidechain, α-aminoacid β-aminoacid, linker, ormodulator may comprise a reporter. The reporter can be an affinityreporter, colored reporter, fluorescent reporter, magnetic reporter,radioisotopic reporter, peptide reporter, metal reporter, nucleic acidreporter, lipid reporter, glycosylation reporter, reactive reporter,enzyme inhibitor, biomolecular substrate, and the like, or a precursorto any of such reporters.

In a further detailed embodiment, the steps of the inventive method areenzyme-catalzyed. Phosphorylation of the labeled pantetheine orderivative thereof can be catalyzed by a kinase such as pantothenatekinase. Adenylation of the labeled phosphopantetheine or derivativethereof can be catalyzed by an adenylyltransferase such asphosphopantetheine adenylyltransferase. Phosphorylation of the3′-hydroxyl of the labeled dephosphoCoenzyme A or derivative thereof canbe catalyzed by dephospho-CoA kinase.

The methods of the present invention find wide application incharacterizing biochemical pathways as well as characterizing proteinexpression, activity, or function in a cell. Accordingly, the analogsgenerated by the methods of the invention may be used to identifyproteins, isolate proteins, assay for the expression and/or activity ofproteins, screen for proteins, quantify temporal events related to theexpression of proteins, assay for the function of proteins, detect thelocation of proteins within a cell, inhibit proteins, activate proteins,examine the structure of proteins, assay for regulation of proteins,identify a cell, cell line, organism, or class of organisms based on thepresence or absence of a protein, or determine the virulence level of acell or organism. The analogs that may be used for these purposesinclude without limitation, the labeled pantetheine or derivativesthereof, the phosphopantetheine analogs and derivatives thereof, thelabeled dephosphoCoenzyme A analogs and derivatives thereof, and thelabeled coenzyme A analogs and derivatives thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the pantetheine analogs (1A-1C) withrepresentation of exemplary modules.

FIG. 2 shows in vivo metabolic labeling of a carrier protein (CP) viacellular uptake of an exemplary pantetheine analog (1A1) and conversionto CoA analog (4) by CoaA, CoaD, and CoaE.

FIGS. 3 a and 3 b shows in vitro enzymatic reconstitution of themetabolic-labeling process.

FIGS. 4 a, 4 b and 4 c shows in vivo tagging of carrier protein fusionconstruct within E. coli.

FIG. 5 shows schematic demonstrating synthesis of a pantetheine analog(1A1).

FIG. 6 shows an application of labeled CoA analogs (1A-1C).

FIG. 7 shows structures of pantetheine analogs and biotin detectionagents used in this study.

FIG. 8 shows a general strategy for in vivo labeling of carrier proteinby pantetheine analogs.

FIGS. 9 a, 9 b, 9 c, 9 d and 9 e show in vivo and in vitro activity ofpantetheine analog panel.

FIGS. 10 a, 10 b, 10 c and 10 d show in vitro labeling ofacyl-carrier-protein (ACP).

FIGS. 11 a, 11 b and 11 c show in vivo carrier protein labeling.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare intended to be construed according to their ordinary meaning in theart.

“Coenzyme” refers to a catalytically active, low molecular masscomponent of an enzyme; and also refers to a dissociable, low-molecularmass active group of an enzyme that transfers chemical groups orhydrogen or electrons. Coenzyme A (CoA) is an exemplary coenzyme.Non-natural coenzyme derivatives, for example, non-natural coenzyme Aderivatives, can be synthesized to contain modifications of the naturalCoA molecule with variant moieties at key locations on the molecule. Forinstance, a library of derivatized functionality at backbone carbonswithin the modulator, α-aminoacid or β-aminoacid or linker andω-functionalized amine or sidechain sub-groups of pantetheine can becreated. These derivatives can contain variation within thefunctionality within the pantetheine backbone as given by a reporter (R)as shown in FIG. 1. Modifications about the reporter R can include theappendage of alkyl, alkoxy, aryl, aryloxy, hydroxy, halo, thiol groupsor be an antigen, dye, chromaphore, cofactor, peptide, ketide,polyketol, terpene, ligand, polymer, surface, oligonucleotide,initiator, radiolabel, natural product, biosynthetic intermediates,inhibitor, organometallic complex, photoaffinity reporter.

FIG. 1 shows the structure of the pantetheine analogs (1A-1C) withrepresentation of exemplary modules. Pantetheine is constructed of threemodules. The first module contains an ω-functionalized amine or asidechain. The second module contains either α-aminoacid, linker orβ-aminoacid. The third module contains a modulator. X can be but is notlimited to alkyl chain, reporter (R), cofactor, peptide, ketide,polyketol, terpene, ligand, polymer, site for surface attachment,initiator, oligonucleotide, radiolabel, natural product, biosyntheticintermediates, inhibitor, or organometallic complex. U can but is notlimited to an oxygen, sulfur or two hydrogens. Y can be but is notlimited to N, O, S or P atom or a methylene group, CH2. W can be but isnot limited to an aromatic, alkyl, polyether, polymer, or alkenyl chain.

“Carrier protein domain” refers to a protein sequence obtained from abiosynthetic gene that naturally is modified by CoA or a CoA derivativeby a phosphopantetheinyl transferase (PPTase). This domain can either bea full protein, a complex of proteins, a fusion construct, or a shortpeptide sequence which may be derived from natural biosynthetic genes orsynthesized artificially. Artificial synthetic peptide substrates can becarrier protein domains including, but are not limited to, the11-residue ybbr tag. Yin et al., Proc. Natl. Acad. Sci. U.S.A.102:15815, 2005. Artificial synthetic peptide substrates and derivativesthereof can be used as a protein fusion or as the peptide substrate notfused to another peptide. The carrier protein domain can be labeled withthe reporter that is catalytically transferred from the coenzyme, forexample, coenzyme A.

“Apo-synthase” or “apo-carrier protein” refers to a synthase containinga carrier protein, a carrier protein or a peptide portion of a carrierprotein that contains a serine residue that can be4′-phosphopantetheinylated, but is not 4′-phosphopantetheinylated. Theterm “apo-” denotes a state of protein modification.

“Holo synthase” or “holo-carrier protein” refers to a synthasecontaining a carrier protein, a carrier protein or a peptide portion ofa carrier protein that contains a serine residue that has been4′-phosphopantetheinylated by natural Coenzyme A. The term “holo-”denotes a state of protein modification.

“Crypto-synthase” or “crypto-carrier protein” refers to a synthasecontaining a carrier protein, a carrier protein or a peptide portion ofa carrier protein that contains a serine residue that has been4′-phosphopantetheinylated by a modified derivative of Coenzyme Abearing a reporter. The term “crypto-” denotes a state of proteinmodification.

“Carrier protein-enzyme-coenzyme complex” refers to derivatives ofcoenzyme A labeled with a reporter that transfer the reporter andselectively mark a carrier protein domain. The attachment of thereporter provides a device for selection, identification and/orrecognition of the biosynthetic enzyme. This process arises through theformation of an enzyme-coenzyme complex. Formation of this complex canoccur prior to or after the formation of a complex between the enzymeand its carrier protein substrate. The enzyme-coenzyme complex and/orcarrier protein-enzyme-coenzyme complex is modified by the appendage ofa reporter.

“Target” refers to a molecule that has an affinity for a given reporter.Targets may be naturally occurring or man-made molecules. Also, they canbe employed in their unaltered state or as aggregates with otherspecies. Targets can be attached, covalently or noncovalently, to abinding member, either directly or via a specific binding substance.Examples of targets that can be employed in accordance with thisinvention include, but are not restricted to, antibodies, cell membranereceptors, monoclonal antibodies and antisera reactive with specificantigenic determinants (such as on viruses, cells or other materials),drugs, oligonucleotides, nucleic acids, peptides, cofactors, lectins,sugars, polysaccharides, cells, cellular membranes, and organelles.Targets are sometimes referred to in the art as anti-reporters. As theterm target is used herein, no difference in meaning is intended.Typically, a “reporter-target pair” is formed when two macromoleculeshave combined through molecular recognition to form a complex.

“Library” refers to a collection of individual analogs of coenzymes orreporters. Specificity of individual units of coenzymes and reportersallows identification of specific biosynthetic enzymes within asolution, complex mixture or cell extract.

“Fused construct” refers to known CP domains having a consensus sequencewithin which the post-translational modification takes place. A fusionprotein of the present invention can contain the consensus amino acidsequence or a homologous sequence thereof. The fusion partner can be asshort as 13 amino acids, but it is considered a phosphopantetheinylationsite if it has the consensus pattern. The consensus sequence is thefollowing:[DEQGSTALMKRH]-[LIVMFYSTACHGNQHLIVMFYAGHDNEKHS]-S-[LIVMST]-{PCFY}-[STAGCPQLIVMF]-[LIVMATNHDENQGTAKRHLM]-[LIVMWSTA]-[LIVGSTACRj-x(2)-[LIVMFA];wherein S is the pantetheine attachment site. Concise EncyclopediaBiochemistry, Second Edition, Walter de Gruyter, Berlin New-York (1988);Pugh E. L., et al, J. Biol. Chem. 240: 4727, 1965; Witkowski et al. Eur.J. Biochem. 198: 571, 1991;http://us.expasy.org/cgi-bin/nicedoc.p17PD0000012.

The pattern rules are as follows. The PA (pattern) lines contains thedefinition of a PROSITE pattern. The patterns are described using thefollowing conventions: The standard IUPAC one-letter codes for the aminoacids are used. The symbol V is used for a position where amino acidsubstitutions is accepted. Ambiguities are indicated by listing theacceptable amino acids for a given position, between square parentheses‘[ ]’. For example: [ALT] stands for Ala or Leu or Thr. Ambiguities arealso indicated by listing between a pair of curly brackets ‘ { }’ theamino acids that are not accepted at a given position. For example: {AM}stands for any amino acid except Ala and Met. Each element in a patternis separated from its neighbor by a ‘-’. Repetition of an element of thepattern can be indicated by following that element with a numericalvalue or a numerical range between parenthesis. For example: x(3)corresponds to x-x-x, x(2,4) corresponds to x-x or x-x-x or x-x-x-x.When a pattern is restricted to either the N- or C-terminal of asequence, that pattern either starts with a ‘<’ symbol or respectivelyends with a ‘>’ symbol. In some rare cases (e.g. PS00267 or PS00539),‘>’ can also occur inside square brackets for the C-terminal element.T-[GSTV]-P—R-L-[G>]’ means that either T-[GSTV]-P—R-L-G’ or‘F-[GSTV]-P—R-L>’ are considered. A period ends the pattern.

One aspect of the invention features methods generate analogs ofcoenzyme A, comprising reacting pantetheine or a derivative thereof witha reporter, phosphorylating the labeled pantetheine or derivativethereof to form labeled phosphopantetheine or derivative thereofpantetheine, adenylating the labeled phosphopantetheine or derivativethereof to form labeled dephosphoCoenzyme A, or a derivative thereof,phosphorylating the 3′-hydroxyl of the labeled dephosphoCoenzyme A orderivative thereof to form a labeled coenzyme analog or derivativethereof.

In a preferred embodiment, the pantetheine or derivative of pantetheinecomprises three modules: (1) modulator, (2) α-aminoacid or β-aminoacidor linker and (3) w-functionalized amine or sidechain. Examples of theω-functionalized amine or sidechain include without limitation3,3-dimethyl-1,2-diaminobutane, 1-phenyl-1,2-ethanediamine,3-phenyl-1,2-propanediamine, 3-aminoalanine, 1,2-propanediamine,1,2-butanediamine, 1-amino-2-propanol, 2-amino-1-phenylethanol,α-(aminomethyl)-benzeneethanol, propaneamine, butaneamine, hexaneamine,phenethylamine, 1-amino-3-propanol, 1-amino-3-chloropropane,1-amino-3-bromopropane, 1,2-pentanediamine where Y═N or 1-propanol,1-butanol, 1-hexanol, 1-phenethanol where Y═O. Examples of α-aminoacidinclude without limitation tryptophan, lysine, methionine, phenylalaine,threonine, valine, leucine, isoleucine, arginine, tyrosine, glycine,serine, glutamic acid, aspartic acid, taurine, histidine, proline,alanine, ornithine, aminobutyric acid, aminohexanoic acid,aminoisobutyric acid, argining, asparagine, aspartic acid, butylglycine,citrulline, cyclohexylalanine, cysteine, diaminobutanoic acid,diaminopropionic acid, glutamic acid, glutamine, homoserine,hydroxyproline, isoleucine, isonipecotic acid, methionine, norleucine,norvaline, penicillamine, phenylalanine, phenylglycine, praline,sarcosine, serine, statine, thienylalanine, threonine, tryptophan,homocitrulline, t-butylglycine, a-fluoroglycine, 3,3,3-trifluoroalanine,2-methylalanine, α,α-diphenylglycine, isovaline, α-methylmethionine,2-amino-2-fluoromethyl-3-(1(3)h-imidazol-4-yl)-propionic acid,2-amino-2-(2,2-dimethyl-benzo[1,3]dioxol-5-ylmethyl)-but-3-ynoic acid,2-amino-2-furan-2-yl-propionic acid, 2-amino-2-methyl-hex-5-enoic acidor 5-iodo-tryptophan. Examples of β-aminoacids include withoutlimitation β-alanine, 2-hydroxy-β-alanine, 2-amino-β-alanine,2-methyl-β-alanine, 2-ethyl-β-alanine, 2-phenyl-β-alanine,3-hydroxy-β-alanine, 3-amino-β-alanine, 3-methyl-β-alanine,3-ethyl-β-alanine, 3-phenyl-β-alanine, 2,2-dimethyl-β-alanine,2,2-diphenyl-β-alanine, 3,3-dimethyl-β-alanine, 3,3-diphenyl-β-alanine,2-isopropyl-β-alanine, 3-isopropyl-β-alanine, 2-t-butyl-β-alanine,3-t-β-alanine, 2-fluoro-β-alanine, 3-fluoro-β-alanine,2,2-difluoro-β-alanine, 3,3-difluoro-β-alanine,2,2,3,3-tetrafluoro-β-alanine, β-aminoacrylic acid, 3-amino-propynoicacid, anthranilic acid, 2-amino-cyclohexanecarboxylic acid,2-amino-cyclopentanecarboxylic acid, 2-amino-cyclopropanecarboxylicacid, 3-amino-oxetane-2-carboxylic acid,4-amino-tetrahydro-furan-3-carboxylic acid, 4-amino-furan-3-carboxylicacid, 2-amino-3,3-dimethyl-cyclopropanecarboxylic acid,2-amino-3,3-difluorocyclopropanecarboxylic acid,pyrrolidine-3-carboxylic acid or piperidine-3-carboxylic acid. Examplesof the linker include without limitation 1-amino-3-butanone,4-amino-3-methyl-2-butanone, 4-amino-3-ethyl-2-butanone,4-amino-3-propyl-2-butanone, 4-amino-3-phenyl-2-butanone,4-amino-2-pentanone, 4-amino-4-phenyl-2-butanone, 4-amino-2-hexanone or4-amino-2-heptanone where U=oxygen, or β-alaninamide,2-hydroxy-β-alaninamide, 2-amino-β-alaninamide, 2-methyl-β-alaninamide,2-ethyl-β-alaninamide, 2-phenyl-β-alaninamide, 3-hydroxy-β-alaninamide,3-amino-β-alaninamide, 3-methyl-β-alaninamide, 3-ethyl-β-alaninamide,3-phenyl-β-alaninamide where U=oxygen; or 1-amino-3-butane,4-amino-3-methyl-2-butane, 4-amino-3-ethyl-2-butane,4-amino-3-propyl-2-butane, 4-amino-3-phenyl-2-butane, 4-amino-2-pentane,4-amino-4-phenyl-2-butane, 4-amino-2-hexane, 4-amino-2-heptane, whereU═H,H; or 1-amino-3-butanthione, 4-amino-3-methyl-2-butanthione,4-amino-3-ethyl-2-butanthione, 4-amino-3-propyl-2-butanthione,4-amino-3-phenyl-2-butanthione, 4-amino-2-pentanthione,4-amino-4-phenyl-2-butanthione, 4-amino-2-hexanthione,4-amino-2-heptanthione. where U═S. The modulator typically contains adihydroxy acid or aminohydroxy acid, and preferably is pantoic acid or ahomolog or derivative or pantoic acid. FIG. 1 depicts the assembly ofthese modules.

The modulator, α-aminoacid, β-aminoacid, linker, ω-functionalized amineor sidechain can comprise a reporter. Non-limiting examples of such areporter include Dansyl, BODIPY, fluoresceine, Texas red,carboxy-X-rhodamine, coumarin, aminocoumarin, oregon green, resorufin,rhodamine green, tetracyclin biotin, mannose, galactose, glucose,methotrexate, FK506.

The phosphorylation of the labeled pantetheine or derivative ofpantetheine may be effectuated by any process suitable in the art tophosphorylate a compound. In a preferred embodiment, the phosphorylationis catalyzed by an enzyme of the enzyme class of kinases. Non-limitingexamples of such kinases include, diacylglycerol kinase, ammonia kinase,beta-adrenergic-receptor kinase, branched-chain-fatty-acid kinase,butyrate kinase, D-ribulokinase, deoxynucleoside kinase,(deoxy)nucleoside-phosphate kinase, dihydrostreptomycin-6-phosphate3′alpha-kinase, diphosphoinositol-pentakisphosphate kinase,farnesyl-diphosphate kinase, galactokinase, glucosamine kinase,glutamate 5-kinase, glutamate 1-kinase, homoserine kinase,inositol-trisphosphate 3-kinase, inositol-hexakisphosphate kinase,undecaprenol kinase, L-arabinokinase, lombricine kinase, mannokinase,myosin-heavy-chain kinase, NAD+kinase, opheline kinase, rhodopsinkinase, pantetheine kinase, phosphatidylinositol 3-kinase,1-phosphatidylinositol-3-phosphate 5-kinase,phosphatidylinositol-4,5-bisphosphate 3-kinase,phosphatidylinositol-4-phosphate 3-kinase, phosphoglycerate kinase,phosphoglycerate kinase (GTP), polynucleotide 5′-hydroxy-kinase,pyridoxal kinase, [pyruvate dehydrogenase (lipoamide)] kinase, selenide,water dikinase, rhodopsin kinase, shikimate kinase, taurocyamine kinase,thiamine-diphosphate kinase, xylulokinase, phosphoprotein phosphatase,protein-tyrosine-phosphatase, protein kinase (CDK/MAK), protein-tyrosinekinase, protein-tyrosine kinase (PTK, not ETK, WZC), protein-tyrosinekinase, protein-tyrosine kinase (PTK, not ETK, WZC), protein-tyrosinekinase, protein-tyrosine kinase (PTK, not ETK, WZC), protein kinase,protein-tyrosine kinase, protein-tyrosine kinase (PTK, not ETK, WZC),protein-tyrosine kinase, protein-tyrosine kinase (PTK, not ETK, WZC),protein-tyrosine kinase, protein-tyrosine kinase (PTK, not ETK, WZC),diacylglycerol kinase, alkylglycerol kinase, 1-phosphofructokinase,2-dehydro-3-deoxygluconokinase, 5-dehydro-2-deoxygluconokinase,5-methyldeoxycytidine-5′-phosphate kinase, S-methyl-5-thioribose kinase,6-phosphofructo-2-kinase, glycerone kinase, acetylglutamate kinase,ceramide kinase, adenosine kinase, adenylate kinase, adenylyl-sulfatekinase, agmatine kinase, alkylglycerone kinase, allose kinase, ammoniakinase, arginine kinase, beta-glucoside kinase,branched-chain-fatty-acid kinase, [3-methyl-2-oxobutanoate dehydrogenase(lipoamide)] kinase, butyrate kinase, caldesmon kinase, carbamatekinase, protein kinase, choline kinase, creatine kinase, cytidylatekinase, D-arabinokinase, D-ribulokinase, deoxyadenosine kinase,(deoxy)adenylate kinase, deoxycytidine kinase, deoxyguanosine kinase,T2-induced deoxynucleotide kinase, thymidine kinase, dephospho-CoAkinase, sphinganine kinase, erythritol kinase, ethanolamine kinase,farnesyl-diphosphate kinase, pyruvate kinase, formate kinase,fructokinase, fucokinase, galactokinase, galacturonokinase, glucokinase,glucosamine kinase, lucuronokinase, glutamate 5-kinase, glyceratekinase, glycerol kinase, guanidinoacetate kinase, guanylate kinase,hamamelose kinase, hexokinase, homoserine kinase, hydroxyethylthiazolekinase, hydroxylysine kinase, hypotaurocyamine kinase, inosine kinase,inositol-tetrakisphosphate 5-kinase, inositol-tetrakisphosphate1-kinase, inositol 3-kinase, undecaprenol kinase, kanamycin kinase,dehydrogluconokinase, L-arabinokinase, L-fuculokinase, L-xylulokinase,tetraacyldisaccharide 4′-kinase, lombricine kinase, mannokinase,mevalonate kinase, Ca2+/calmodulin-dependent protein kinase,acylglycerol kinase, adenylate kinase, myosin-light-chain kinase, NAD+kinase, nucleoside-diphosphate kinase, nucleoside-phosphate kinase,nucleoside-triphosphate-adenylate kinase, opheline kinase, pantothenatekinase, agmatine kinase, 1-phosphatidylinositol 4-kinase,phosphatidylinositol 3-kinase, 6-phosphofructokinase,phosphoglucokinase, phosphoglycerate kinase (GTP),phosphomethylpyrimidine kinase, phosphomevalonate kinase,phosphoribokinase, phosphoribulokinase, phosphorylase kinase,polyphosphate kinase, protamine kinase, protein-histidine pros-kinase,protein-histidine tele-kinase, protein-tyrosine kinase, protein-tyrosinekinase (PTK, not ETK, WZC), protein kinase, protein-tyrosine kinase,protein-tyrosine kinase (PTK, not ETK, WZC), protein kinase, proteinkinase, pseudouridine kinase, diphosphate-purine nucleoside kinase,pyridoxal kinase, pyruvate kinase, pyruvate water dikinase, NADH kinase,rhamnulokinase, riboflavin kinase, [RNA-polymerase]-subunit kinase,[RNA-polymerase]-subunit kinase, ribosylnicotinamide kinase,scyllo-inosamine 4-kinase, sedoheptulokinase, shikimate kinase,sphinganine kinase, streptomycin 6-kinase, streptomycin 3-kinase,streptomycin 6-kinase, tagatose kinase, taurocyamine kinase, thiaminekinase, thiamine-diphosphate kinase, thiamine-phosphate kinase,thymidine kinase, dTMP kinase, dTMP kinase, triokinase, [tyrosine3-monooxygenase]kinase, uridine kinase, xylulokinase, protein kinase(CaMK, MLCK, PhK, SNF, KIN, NIM1, MAPKAP, POLO, CHK, ULK, RSK-2nddomain). In a more preferred embodiment, the phosphorylation iscatalyzed by pantothenate kinase.

The adenylation of the labeled phosphopantetheine or derivative thereofto form a labeled dephosphoCoenzyme A or derivative thereof may beeffectuated by any process suitable in the art to adenylate a compound.In a preferred embodiment, the adenylation is catalyzed by an enzyme. Ina more preferred embodiment, the adenylation is catalyzed be anadenylyltransferase. Non-limiting examples of adenylyltransferasesinclude ATP adenylyltransferase, adenylylsulfate-ammoniaadenylyltransferase, anthranilate adenylyltransferase,glucose-1-phosphate adenylyltransferase,[glutamate-ammonia-ligase]adenylyltransferase, nicotinamide-nucleotideadenylyltransferase, nicotinate-nucleotide adenylyltransferase,phenylalanine adenylyltransferase, ribose-5-phosphateadenylyltransferase, aldose-1-phosphate adenylyltransferase, and sulfateadenylyltransferase. In a still more preferred embodiment, theadenylyltransferase is phosphopantetheine adenylyltransferase.

Alternatively, generation of the labeled dephosphoCoenzyme A orderivative thereof may be accomplished by dephosphorylation catalyzed bythe action of a phosphatase. Such a reaction was recently described bySilva et al., Drug Metabolism Disposition 32: 1304, 2004. Still anotheralternative means to generate the labeled dephosphoCoenzyme A orderivative thereof is to isomerize iso-CoA through cyclic-CoA to CoA, asdescribed by Burns et al., J. Biol. Chem., 280:16550, 2005.

The adenylation step can include modifications within the adenylationreagent, such as adenosine triphosphate, ATP, as shown in FIG. 1. Anyderivative or surrogate of ATP may be used, including withoutlimitation, precursors adenosine diphosphate, ADP, or adenosinemonophosphate, AMP. This can include the use of alternate nucleotidephosphates including thymidine triphosphate, TTP, guanosinetriphosphate, GTP, and cytidine triphosphate, CTP and their biosyntheticprecursors. Modifications can include AMP, ADP, and ATP derivatives thatbear modifications within the carbohydrate side chain, for instance2-deoxyriboses or 2-aminoriboses or 2-fluororiboses or modificationswithin the adenine base including the application of adenine mimics andN- and C-functionalized adenines such as tubercidin triphosphate,8-amino-adenosine triphosphate, 7-leazanebularin, formycin triphosphatc,5′-adenylylmethylene-diphosphonate, N-6-(benzyl)-ATP,2-[(2-nitrophenyl)amino]ethyl triphosphate, 2-(phenylamino)ethyltriphosphate, adenosine 5′-o-(3-thio)triphosphate, 2-methylthio-AMP,2-chloroadenosine, 2-chloro-5′-adenylylmethylenediphosphonate or otherrelated nucleotide mimics.

The phosphorylation of the 3′-hydroxyl of the adenylated labeleddephosphoCoenzyme A or derivative thereof to form a labeled coenzyme Aanalog or derivative thereof may be effectuated by any process suitablein the art to phosphorylate a compound. In a preferred embodiment, thephosphorylation is catalyzed by an enzyme of the enzyme class ofkinases. In a more preferred embodiment, the phosphorylation iscatalyzed by dephospho-CoA kinase.

The labeled coenzyme A analog or derivative thereof may be furtherreacted with a carrier protein domain to form a labeled protein. Thelabeling reaction may be effectuated by any process suitable in the artto catalyze the transfer of the reporter to the protein. In a preferredembodiment, the labeling reaction is catalyzed by an enzyme. In a morepreferred embodiment the labeling reaction is catalyzed by aphosphotransferase. In a still more preferred embodiment, the labelingreaction is catalyzed by 4′-phosphopanthetheinyltransferase.

The labeling-process may transfer a diversity of reporters, includingorganic or inorganic molecules, or biomolecules, or any fragment,analog, homolog, derivative, or conjugate thereof. Biomolecules includenucleic acids, polypeptides, polysaccharides, lipids, and the like. Morespecifically, the reporter may be, without limitation, an affinityreporter, colored reporter, fluorescent reporter, magnetic reporter,radioisotopic reporter, peptide reporter, metal reporter, nucleic acidreporter, lipid reporter, glycosylation reporter, reactive reporter,enzyme inhibitor, substrate for a biomolecule, or the like. Similarly,the reporter may be any precursor to any of the above-mentionedreporters including, but not limited to, bioorthogonal chemical labelssuch as ketone, azide, alkyne, or triarylphosphine analogs.

The reporter may be chosen as substrate or mimetic for study ofbiomolecular structure and function. For this application, the reportersfunction can be derived from molecules generated at the state ofphosphopantetheine e using analogs such as 2A-2C, dephosphoCoA throughanalogs such as 3A-3C, CoA through analogs such as 4A-4C andcrypto-carrier protein through analogs such as 5A-5C. This reporter cantherefore be used to assist structural analyses for instance throughnuclear magenetic resonance (NMR) spectroscopy, X-ray crystallographicanalysis, and fluorescent methods. The reporter can also be used toguide drug design, to act as a drug or drug precursor, to act as acomponent of an assay. Another aspect of the present invention featuresmethods to generate analogs of coenzyme A in a cell. Such methodscomprise reacting pantetheine or a derivative thereof with a reporter,contacting the cell with the labeled pantetheine or derivative ofpantetheine such that the labeled pantetheine or derivative orpantetheine enters the cell, phosphorylating the labeled pantetheine orderivative thereof to form labeled phosphopantetheine or derivativethereof pantetheine, adenylating the labeled phosphopantetheine orderivative thereof to form labeled dephosphoCoenzyme A, or a derivativethereof, phosphorylating the 3′-hydroxyl of the labeleddephosphoCoenzyme A or derivative thereof to form a labeled coenzymeanalog or derivative thereof.

The cell may be any eukaryotic, prokaryotic, or archaebacterial cell.The cell may be contacted with the labeled pantetheine or derivative ofpantetheine by any means suitable in the art. Numerous means to deliversmall molecules to cells are known and established in the art.Non-limiting examples of such means include liposomes, microinjection,electroporation, biolistics, receptor mediated endocytosis, cellpenetrating peptides, and the like. The skilled artisan will understandthat delivery vehicles can vary with the cell type, and will similarlyappreciate that specialized delivery vehicles and methods are availablefor delivery of small molecules to specific cells or tissues. In apreferred embodiment, the labeled pantetheine or derivative ofpantetheine is formulated as part of the cell growth medium, anddelivered to the cell by natural uptake.

The methods of the present invention may be used in a variety ofapplications. Non-limiting examples of such applications includepreparation of fluorescent proteins in vitro and in vivo without theneed to genetically engineer such proteins, as occurs with greenfluorescent protein, studying biochemical processes of the cell, markingof cell signaling pathways, characterization of the cell cycle,elucidation of protein-ligand interaction, characterization of cell-cellinteractions, determination of whether a cell is viable or dead,profiling cell structures, staining of cellular organelles,identification of cell type, modification of cellular function,regulation of cell differentiation, isolation of cells, profiling andidentification of microbes, isolation of microbes, evaluation ofembryology and developmental biology, detection of disease or diseasestate, evaluation of clinical samples, preparation of bioassays, proteinfunction analysis, protein structure analysis, metabolic regulation,metabolic analysis, drug delivery, drug localization, natural productbiosynthetic identification, natural product biosynthetic elucidation,natural product biosynthetic manipulation, metabolic engineering, drugscreening, drug development, as a drug, and the like.

The following examples are provided to illustrate the invention ingreater detail. The examples are intended to illustrate, not to limit,the invention.

EXEMPLARY EMBODIMENTS Example 1 Pantetheine Analog Synthesis

All reactions were carried out under an atmosphere of argon inflame-dried glassware with magnetic stirring. Column chromatography wasperformed on 0.25 mm silica gel 60-F plates. Visualization wasaccomplished with UV light, o-tolidine, cerium molybdate, or ninhydrindips followed by heating. ¹HNMR and spectra were recorded on a 500, 400,and 300 MHz spectrometer at ambient temperature. Data were reported asfollows: chemical shifts in parts per million (δ, ppm) from an internalstandard [deuterated methanol (CD₃OD), deuterated chloroform (CDCl₃), ordeuterated dimethyl sulfoxide (DMSO)], multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, and m=multiplet), integration, andcoupling constant (Hz). ¹³CNMR were recorded on a 500, 400, and 300 MHzNMR at ambient temperature. Chemical shifts are reported in ppm fromCD₃OD, CDCl₃, or DMSO. High resolution mass spectrometry was obtainedusing fast atom bombardment (FAB) as the ion source. The matrix in allcases was 3-nitrobenzyl alcohol and the reference was polyethyleneglycol.

Synthesis of3-{[2-(4-Methoxy-phenyl)-5,5-dimethyl-[1,3]dioxane-4-carbonyl]-amino}-propionicacid (8A1)

Pantothenic acid (7) (4.6 g, 2.24×10⁻² mols) was dissolved in dry DCM(30 mL). Camphor sulfonic acid (0.52 g, 2.24×10⁻³ mols) andp-anisaldehyde-dimethoxy-acetal (3.82 mL, 2.24×10⁻² mols) were added tothe reaction mixture. The reaction was stirred overnight at roomtemperature with a drying tube. The crude reaction product wasconcentrated and purified silica gel chromatography (6:1hexane:EtOAC-EtOAc) to yield a white solid (8A1) (3.8 g, 51% yield).mp=135-136° C. ¹HNMR (400 MHz, DMSO) 0.93 (s, 3H), 0.99 (s, 3H), 2.38(t, 2H, J=6.8 Hz), 3.25 (m, 1H), 3.34 (m, 1H), 3.59 (d, 1H, J=10.8 Hz),3.62 (d, H, J=10.8 Hz), 3.74 (s, 3H), 4.07 (s, 1H), 5.50 (s, 1H), 6.91(d, 2H, J=8.8 Hz), 7.41 (d, 2H, J=8.8 Hz). ¹³C-NMR (400 Mz, DMSO) 19.7,22.2, 33.2, 34.4, 34.9, 55.8, 78.0, 83.8, 101.1, 114.0, 128.4, 131.1,160.3, 168.9, 173.8. IR (NaCl, thin film), 3420, 2959, 1729, 1654, 1617,1540, 1520, 1251, 1105 cm⁻¹. MS (ESI) [M+Na]⁺360.1. HRMS (FAB) (m/z):[M+H]⁺ calcd for C₁₇H₂₃O₆N, 338.1598, found 338.1594.

Synthesis of 2-(4-Methoxy-phenyl)-5,5-dimethyl-[1,3]dioxane-4-carboxylicacid(2-{6-[2-(7-dimethylamino-2-oxo-2H-chromen-4-yl)-acetylamino]-hexylcarbamoyl}-ethyl)-amide(9A1)

Protected pantothenic acid 8A1 (0.434 g, 1.34×10⁻³ mol) was dissolved in30 mL of DMF. EDC (0.529 g, 2.68×10⁻³ mol), HOBT (0.411 g, 2.68×10⁻³mol), and triethylamine (0.374 mL, 2.68×10⁻³ mol) were added to thereaction mixture. ω-Aminoacid 6A1 (0.600 g, 1.34×10⁻³ mol) was dissolvedin 5 mL of DMF and added to the reaction mixture. The reaction stirredover night at room temperature under Ar before being evaporated to yielda yellow oil. The oil was redissolved in 50 mL of DCM and the organiclayer washed with 15% citric acid (3×50 mL), saturated NaHCO₃ (3×50 mL),water (2×50 mL), and brine (2×50 mL) and dried with sodium sulfate. Theconcentrated crude product was purified by silica gel chromatography(DCM: 10% MeOH:DCM) to yield a pale yellow solid 9A1 (0.680 g, 77%yield). ¹ HNMR (400 MHz, CDCl₃) δ 1.04 (s, 3H), 1.06 (s, 3H), 1.21 (m,4H), 1.39 (m, 4H), 2.38 (t, 2H, J=6.4 Hz), 3.00 (s, 6H), 3.12 (m, 4H),3.45 (m, 2H), 3.55 (s, 2H), 3.61 (d, 1H, J=11.2 Hz), 3.66 (d, 1H, J=11.2Hz), 3.76 (s, 3H), 4.02 (s, 1H), 5.42 (s, 1H), 6.00 (s, 1H), 6.39 (d,1H, J=2.4 Hz), 6.48 (t, 1H, J=4.8 Hz, —NH), 6.55 (dd, 1H, J=8.8, 2.4Hz), 6.68 (t, 1H, J=5.2 Hz, NH), 6.86 (d, 2H, J=8.8 Hz), 7.09 (t, 1H,J=6.0 Hz, NH), 7.39 (d, 2H, J=8.8 Hz), 7.46 (d, 1H, J=8.8 Hz). ¹³CNMR(400 Mz, CDCl₃) 19.4, 22.1, 26.2, 29.3, 29.5, 33.3, 35.3, 36.2, 39.2,39.6, 40.3, 40.6, 55.5, 78.6, 84.0, 98.2, 101.5, 108.7, 109.4, 110.0,113.9, 126.1, 127.7, 130.3, 150.8, 153.2, 156.1, 160.4, 162.3, 168.4,169.7, 171.2. IR (NaCl, thin film), 3320, 2930, 2860, 1707, 1652, 1616,1531, 1403 cm⁻¹. MS (ESI) [M+Na]⁺687.40. HRMS (FAB) (m/z): [M+H]⁺ calcdfor C₃₆H₄₈O₈N₄, 665.3545, found 665.3542.

Synthesis of Labeled-Coa Analog (1A1)

Compound 9A1 (0.680 g, 1.0×10⁻³ mol) was dissolved in 50:50 l M HCl:THFand the reaction stirred at room temperature for 2 h. AG-1-X8 StrongBasic anionic exchange resin was added to the reaction mixture until thesolution was neutral. The crude product was concentrated under vacuumand redissolved in methanol (50 mL) and washed with hexanes (3×, mL).The crude product was again concentrated and purified by silica gelchromatography (DCM: 10% MeOH:DCM) to yield pure pale yellow solid 1A1(0.529 g, 95%) with a melting point, mp=135-136° C. Compound 1A1 is anexample of pantetheine analog type 1A. 1HNMR (400 MHz, CD3OD) δ 0.80 (s,3H), 1.20 (m, 4H), 1.35 (m, 2H), 1.40 (m, 2H), 2.29 (t, 2H, J=6.4 Hz),3.00 (s, 6H), 3.01 (m, 2H), 3.10 (t, 2H, J=6.8 Hz), 3.32 (m, 4H), 3.56(s, 2H), 3.77 (s, 1H), 5.93 (s, 1H), 6.43 (d, 1H, J=2.8 Hz), 6.63 (dd,1H, J=9.2, 2.8 Hz), 7.45 (d, 2H, J=9.2 Hz). ¹³CNMR (400 Mz, CDCl₃) 20.9,21.4, 27.5, 27.6, 30.2, 30.3, 36.4, 36.5, 40.3, 40.4, 40.5, 70.3, 77.1,98.6, 109.6, 110.2, 110.4, 126.7, 152.8, 154.5, 156.9, 164.0, 170.7,173.3, 175.7. IR (NaCl, thin film), 3488, 3310, 2917, 1712, 1634, 1606,1542 cm−1. MS (ESI) [M+Na]+569.32. HRMS (FAB) (m/z): [M+H]+calcd forC28H42O7N4, 547.3126, found 547.3121.

Example 2 In Vivo Labeling and Cellular Uptake Studies

For the in vivo carrier protein labeling studies, E. coli (BL-21) weretransformed by electroporation with plasmids encoding the genes for VibB(V. cholerae) and Sfp (B. subtilis). Cultures were grown to OD₆₀₀=0.6and then induced with 1 mM IPTG. At the same time differingconcentrations of 7-dimethylaminocoumarinacetic acid labeled-pantetheinewere added. The cultures were grown up to 16 h with time points taken atintervals to investigate uptake and labeling. The cells were centrifugedand resuspended in lysis buffer (50 mM sodium phosphate, 300 mM NaCl),washed three times in an equal volume of lysis buffer, and then lysed byincubation with lysozyme (1 mg/ml) for 1 h on ice followed bysonication. Lysates were then examined for fluorescence (excitation at360 nm; emission at 465 nm) and run on 12% PAGE for visualization. Gelswere imaged using conventional CCD imaging.

Example 3

Assessment of Effect of the Labeled-Pantetheine on E. coli Growth

To assess the effect of the labeled-pantetheine on E. coli, cultures ofBL-21. E. coli were grown with increasing concentrations of thecompound. Culture tubes containing 5 ml of LB were inoculated with 1 ulof E. coli from overnight growths. At the same time thelabeled-pantetheine was added. Growth was measured by monitoring theOD₆₀₀ of the cultures over time.

Example 4 In Vitro Synthesis of Labeled-CoA and HPLC Analysis

The conversion of labeled-pantetheine to labeled-CoA was achieved invitro by incubation of the pantetheine analogue with the enzymes CoaA,CoaD, and CoaE from E. coli. Reactions were run in 20 mM KCl, 5 mM 10 mMMgCl₂, and 50 mM Tris-Cl pH 7.5. CoaA (4 uM), CoaD (15 uM), and CoaE (30mM) were then added and the reactions were incubated at room temperaturefor 1 h. Progress of the reactions was analyzed using reverse-phase C18column equilibrated in 100% solution A (0.05% aqueous TFA). Productswere eluted using a gradient with solution B (0.05% aqueous TFA) at aflow rate of 0.9 mL/min The method used began with an isocratic stepfrom 0 to 5 min at 100% solution A, followed by an increasing gradientwith solution B until at 25 min the solvent composition was 50% SolventA and 50% Solvent B. The in vitro synthesis reactions of labeled-CoAwere also visualized by PAGE. The carrier protein VibB and PPTase Sfpwere added to the reaction mix, incubated for 30 min at 37° C. and thenrun on 12% PAGE. Gels were documented using conventional CCD imaging.

Example 5 Tagging Heterologously Expressed Carrier Protein Domains

Fluorescent tagging with derivatives was repetitively conducted onproteins from crude cell lysate from recombinant E. coli BL21 cellsexpressing a carrier protein (i.e., VibB). Cell lysate was dialyzed toremove small molecules (<3 or <10 kDa), incubated with CoA-DYE andrecombinant Sfp, and analyzed by SDS-PAGE. When viewed underirradiation, recombinant VibB is visualized as a fluorescent band thatwas verified with two methods. First, standard Coomasie staining showedthe fluorescent band to have the proper molecular weight when comparedto molecular weight markers. Second, an identical gel waselectrophoretically transferred to a polyvinylidene fluoride (PVDF)membrane, and the fluorescent band was excised from the membrane. Thismembrane piece was subjected to N-terminal amino acid sequencing byEdman degradation. The first 10 amino acids of the returned sequence,MAIPKIASYP, mapped to the correct protein, VibB, when searched withBLAST against 1.4 million sequences in the GenBank. Broad applicabilityof these techniques is anticipated for validating proper folding andmodification ability of recombinant PK and NRP systems.

One liter of E. coli BL21 (de3) cells, grown using standard methods ofIPTG induced overexpression of recombinant proteins, were lysed bysonication at 0° C. in 30 ml of 0.1 M Tris-Cl pH 8.0 with 1% glycerol inthe presence of 500 μL of a 10 mM phenylmethanesulfonyl fluoride (PMSF)solution in isopropanol with 50 μL of a protease inhibitor cocktail (amixture of protease inhibitors with broad specificity for the inhibitionof serine, cysteine, aspartic and metallo-proteases, and aminopeptidasescontaining 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatinA, E-64, bestatin, and sodium EDTA. After centrifugation at 4000×g for10 min, 200 μL of this cell lysate was treated with 80 μL of thelabeled-CoA solution and 1 μl. (30 μg) of 30 mg/mL purified Sfp, and thereaction was incubated at room temperature for 30 min in darkness. An800 aliquot of a 10% trichloroacetic acid solution was added and cooledat −20° C. for 30-60 min. The samples are centrifuged for 4 min, and thesupernatant was removed. The pellets are resuspended in 1:1 mixture of1.0 M Tris-HCl pH 6.8 and 2×SDS-PAGE sample buffer (100 mM Tris-Cl pH6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue). This solution isplaced in boiling water for 5-10 min and separated using SDS-PAGEelectrophoresis on a 12% Tris-Glycine. Tagged proteins are visualized bytrans-illumination and the resulting images captured with CCD camera.The fluorescent bands originate from crypto-synthases.

Example 6 Tagging of Purified Recombinant Carrier Protein Domains

Fluorescently-labeled CoA were prepared by selective modification of thefree thiol of coenzyme A. This labeled-CoA derivative was then incubatedwith heterologously expressed and purified Sfp and VibB, a small proteinfrom the Vibrio cholera vibriobactin biosynthetic machinery containingonly one carrier protein domain. Analysis was performed with SDS-PAGE,and a single fluorescent band was visualized by eye using theappropriate wavelength of light for excitation. The excitationwavelength was chosen based on using the appropriate combination ofexcitation with UV-visible light and the appropriate cutoff filters.Coomasie staining of the gel verified the fluorescent reporter to becrypto-VibB.

This example demonstrates the utility of this method to fluorescentlytag purified over-expressed and purified VibB, a standalone CP domain.In this example, VibB, a 32.6 kDa protein, is fluorescently-tagged.Tagging was conducted by the addition of a biotin-tagged derivative anda PPTase such as Sfp. SDS-page electrophoresis was used to separateproteins.

Recombinant His-tagged VibB, purified by nickel chromatography, wasdialysed to a 0.6 mg/ml solution in 0.1M TRIS-HCl, pH 8.4 with 1%glycerol. A 200 μL aliquot of this solution is treated with 80 μL of thelabeled-CoA solution (see Preparation of modified CoA derivatives). Thereaction is incubated at room temperature for 30 min in darkness. A 50μl, aliquot of a 10 mg/mL solution of bovine serum albumin (BSA) isadded, and the protein is precipitated by the addition 800 μL of a 10%trichloroacetic acid solution and cooling at −20° C. for 30-60 min. Thesamples are centrifuged at 13,000×g for 4 min, and the supernatant isremoved. The pellet was resuspended in 1:1 mixture of 1.0 M Tris-HCl pH6.8 and 2×SDS-PAGE sample buffer (100 mM Tris-Cl pH 6.8, 4% SDS, 20%glycerol, 0.02% bromophenol blue). This solution was placed in boilingwater for 5-10 min and separated using SDS-PAGE electrophoresis on a 12%Tris-Glycine. Tagged proteins were visualized by trans-illumination andthe resulting images captured with CCD camera.

Example 7 Tagging of Natively Expressed Carrier Protein Domains

Fluorescent tagging was repeated on proteins from crude cell lysate fromrecombinant E. coli K12 cells following iron-starving conditions, whichinclude growth in minimal nutrient media and iron chelation by growth inminimal media and addition of 2,2-dipyridyl. Reagents used for thisfluorescent tagging are shown in FIG. 2. These conditions induceenterobactin production in the organism, which is synthesized by NRPsynthase proteins EntB, EntE, and EntF. Both EntB and EntF containcarrier protein domains that can be post-translationally modified by4′-phosphopantetheinyltransferase. Cell lysate from the iron starvedcells was dialyzed to remove small molecules (<10 kDa), incubated withCoA-DYE and recombinant Sfp, and analyzed by SDS-PAGE. When viewed underirradiation, recombinant EntF and EntB are visualized as fluorescentbands that can be verified with two methods. First, standard Coomasiestaining showed the fluorescent bands to have the proper molecularweight when compared to molecular weight markers. Second, bands from anunstained gel were subjected to mass spectroscopic protein sequencing(Qstar MS-MS) to reveal the sequences of EntF and EntB after searchingGenBank protein databank.

FIG. 2 shows in vivo metabolic labeling of a carrier protein (CP) viacellular uptake of an exemplary pantetheine analog (1A1) and conversionto CoA analog (4) by CoaA, CoaD, and CoaE. This process is followed byreaction of a PPTase with (4) and a carrier protein yielding labeledprotein (5). This example depicts VibB, a natural fusion constructcomprised of a carrier protein fused to an isochorismate lyase (ICL).The gray circle denotes a reporter.

FIG. 3 shows in vitro enzymatic reconstitution of the metabolic-labelingprocess. (a) HPLC analysis of the stepwise conversion of (1A1) toreporter-labeled CoA analog (4). (b) SDS-PAGE gel depicting the labelingof a carrier protein was used, by 4 with PPTase. For this example, thecarrier protein was VibB, a protein in the vibriobactin synthase fromVibriobacter cholerae, and the PPTase was Sfp, surfactin PPTase fromBacillus subtilis. A fluorescent reporter was used, and the labeledcarrier protein, VibB, was detected by fluorescence imaging.Intermediates (2) and (3) can also be visualized through gel analysis.

FIG. 4 shows in vivo tagging of carrier protein fusion construct withinE. coli. (a) Growth of E. coli culture (OD₆₀₀) over a range inconcentrations of (1A1). (b) In vivo formation of crypto-CP statefollowing a time course after addition of 1 mM (1A1) to culture. (c) Invivo formation of crypto-CP state following a time course followingaddition of 100 μM (1) to culture.

FIG. 5 shows schematic demonstrating synthesis of a pantetheine analog(1A1).

FIG. 6 shows an application of labeled CoA analogs (1A-1C). Theseanalogs can either be used directly for biomolecular identification,analysis, or screening or they can be conjugated to a carrier protein(CP) and then be used to biomolecular identification, analysis, orscreening. A combination of any of these and related methods can bedeveloped from 1A-1C.

E. coli K12 cells are starved of iron as follows. E. coli K12 cells in a1 liter of Lauria-Bertani (LB) media was incubated at 37° C. to an OD of−0.7. The cells are treated with 2,2-dipyridyl to a final concentrationof 0.2 mM and allowed to incubate an additional 4 h at 37° C. Theculture was then centrifuged, and the resuspended cell pellets werelysed by sonication at 0° C. in 30 ml of 0.1 M Tris-Cl pH 8.0 with 1%glycerol in the presence of 500 μL of a 10 mM phenylmethanesulfonylfluoride (PMSF) solution in isopropanol with 50 μL of a proteaseinhibitor cocktail: a mixture of protease inhibitors with broadspecificity for the inhibition of serine, cysteine, aspartic andmetallo-proteases, and aminopeptidases, including4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatin A, E-64,bestatin, and sodium EDTA, Sigma-Aldrich Inc.) An 80 μL aliquot of themodified-CoA solution was added to 200 μL of the cell lysate along with30 ug of 30 mg/mL purified Sfp. The resulting mixture was incubated atroom temperature for 30 min in darkness. Proteins were precipitated fromthis solution by the addition of 800 μL of a 10% trichloroacetic acidsolution and cooling at −20° C. for 30-60 min. The samples werecentrifuged at 4000×g for 4 min, and the supernatant is removed. Thepellet was resuspended in 1:1 mixture of 1.0 M Tris-HCl pH 6.8 and2×SDS-PAGE sample buffer (100 mM Tris-Cl pH 6.8, 4% SDS, 20% glycerol,0.02% bromophenol blue). This solution was placed in boiling water for5-10 min and separated using SDS-PAGE electrophoresis on a 12%Tris-Glycine. Tagged proteins were visualized by trans-illumination andthe resulting images captured with CCD camera. Blotting analysis wasconducted using the biotinylated-CoA derivative as described below.

Use of this method to identify proteins containing at least one CPdomain within the cell lysate of native producer organism has beencompleted as another example. In this example, EntB, a 32.6 kDa protein,was selectively tagged within the culture of its natural host (E. coli).SDS-page electrophoresis was used to separate proteins. Tagging wasconducted by the addition of a fluorescently-tagged derivative as givenby a PPTase such as the Bacillus subtilis Sfp transferase. Fluorescenttagging was identified as a fluorescent band using conventional CCDimage analysis.

Example 8 SDS-Page Electrophoresis

SDS-page electrophoresis was used to detect polyketide, non-ribosomalpeptide, and fatty acid synthases continuing carrier proteins throughprotein tagging with CoA-labeled by a fluorescent dye, biotin, acarbohydrate or oligosaccharide, a peptide sequence, or anotherselectable moiety. Proteins from natural or engineered organisms weretagged with the use of a 4′-phosphopantetheinyltransferase and the CoAderivative, and subsequently separated by SDS-PAGE. The separatedproteins were visible in the gel at this stage (as in the case offluorescent tagging), or the gel can be further processed to allowvisualization of the tagged proteins. Visualized pieces of the gel canbe excised for protease digestion and analysis, protein sequencing viaEdman degradation or mass spectrophotometric techniques, or extractedfor solution-phase assays of the purified proteins. The whole gel canalso be subjected to electrophoretic transfer of the proteins to amembrane or other substrate for blot analysis.

Example 9 Native Protein Polyacrylamide Gel Electrophoresis

This technique was used to detect PK, NRP, and fatty acid synthasescontinuing carrier proteins via native protein gel electrophoresisthrough protein tagging with CoA-labeled by a fluorescent dye, biotin, acarbohydrate or oligosaccharide, a peptide sequence, or anotherselectable moiety. Proteins from natural or engineered organisms weretagged with the use of a 4′-phosphopantetheinyltransferase and the CoAderivative, and subsequently separated by a native proteinpolyacrylamide gel. The separated proteins were visible in the gel atthis stage (as in the case of fluorescent tagging), or the gel can befurther processed to allow visualization of the tagged proteins.Visualized pieces of the gel can be excised for protease digestion andanalysis, protein sequencing via Edman degradation or massspectrophotometric techniques, or extracted for solution-phase assays ofthe purified proteins. The whole gel can also be subjected toelectrophoretic transfer of the proteins to a membrane or othersubstrate for blot analysis.

Example 10 Blot Analysis

Blotting was performed to identify proteins with carrier proteindomains. It was found that PPTases such as Sfp would accept a variety ofCoA derivatives for transfer onto a carrier protein, including a biotintag, which could be visualized by electroblotting onto nitrocellulosefollowed by binding with streptavidin that is modified forvisualization. Biotin-CoA derivative was synthesized using a variety oflinked biotin tags using a method comparable to that to attachreporters. The biotin-linked 4′-phosphopantetheine was successfullytransferred to apo-VibB with recombinant Sfp. The biotin-tagged VibB wasthen identified by a blot: purified with SDS-PAGE or native protein gel,electro-transferred to nitrocellulose, and incubated sequentially withstreptavidin-linked alkaline phosphatase and 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT). The biotin-labeled VibBprotein on the nitrocellulose membrane stained dark blue due toenzymatic dephosphorylation of BICP and precipitation of the dark blueproduct through oxidation by NBT. This assay provides convincingevidence that a biotin-streptavidin technique can also be used to purifyPK and NRP synthases that contain carrier protein domains with affinitychromatography. This assay can be conducted with any affinity tag andmolecular binding partner, including mannose-concanavalin A andpeptide-antibody interactions. These results have been reproduced usingmannose-linked CoA tagging to VibB with Sfp, separating on SDS-PAGE,blotting to nitrocellulose, and visualizing with concanavalin-linkedperoxidase and peroxidase substrate (3-Amino-9-ethylcarbazole).

One liter of E. coli BL21 (DE3) cells induced to express recombinantVibB protein were lysed in 30 mL 1M Tris-Cl pH 8.0 with 1% glycerol inthe presence of 500 μL of a 10 mM phenylmethanesulfonyl fluoride (PMSF)solution in isopropanol by sonication. A 50 μL of a protease inhibitorcocktail mixture containing protease inhibitors with broad specificityfor the inhibition of serine, cysteine, aspartic and metallo-proteases,and aminopeptidases including 4-(2-aminoethyl)benzenesulfonyl fluoride(AEBSF), pepstatin A, E-64, bestatin, and sodium EDTA was added to thissample prior to lysis. A 40 μL of the biotinylated-CoA analog solutionwas added to 200 μL of cell lysate containing overexpressed VibB and 1μL of a 34 mg/mL solution of purified Sfp and the reaction was incubatedat room temperature for 30 min in darkness.

Proteins were precipitated from this solution by the addition of 800 μLof a 10% trichloroacetic acid solution and cooling at −20° C. for 30-60min. The samples were centrifuged at 14000×g for 4 min, and thesupernatant was removed. The pellet was resuspended in 1:1 mixture of1.0 M Tris-HCl pH 6.8 and 2×SDS-PAGE sample buffer (100 mM Tris-Cl pH6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue). This solution wasplaced in boiling water for 5-10 min and separated using SDS-PAGEelectrophoresis on a 12% Tris-Glycine. Following separation, the gel wastransferred to nitrocellulose and blotted.

Blots were incubated with 5% milk in TBST for 30 min at room temperaturewith shaking. The blots were then transferred directly to 1 mL of a 5%milk in TBST solution containing 1 μL of 25 mg/mL streptavidin-alkalinephosphatase conjugate and incubated at room temperature for 1 hour.After this incubation, the blot was washed 3 times for 10 min with 20 mLof TBST at room temperature. Finally, the blot was incubated in 2 mL ofAlkaline-phosphatase substrate solution (0.15 mg/mL EOT, 0.30 mg/mL NBT,100 mM Tris, 5 mM MgCl₂ pH 9.5) for 5 min or less at 37° C.

In this example, recombinant VibB has been selected using an affinitymethod. Tagging was conducted by the addition of a biotinylatedCoA-derivative and a PPTase such as the Bacillus subtilis Sfptransferase. Each reaction contained 200 μL of an E. coli lysatecontaining approximately 0.12 μg of VibB. This blot was developed bytransferring protein from a SDS-page gel onto PDVF and/or anitrocellulose paper and developing by the sequential addition of aStreptavidin Alkaline Phosphatase conjugate followed by exposure toBCIP/NBT. The net protein content of the solution was stained byCoomassie blue. A gradient of biotinylated-CoA derivative was placedacross the gel. Metal induction is required for the overexpression ofthe native EntB and EntF proteins to minimize inference when examiningthe overexpression of recombinant carrier proteins conventional E. coliexpression vectors.

Example 11 Affinity Chromatography

Biotinylated CoA derivatives were incubated with crude cell lysate fromVibB-producing E. coli (as described above) and the mixture was run overa small column loaded with streptavidin-linked-agarose resin. Followingwashing, some of the resin was boiled to release biotin-bound protein,and the sample was subjected to SDS-PAGE as well as blotting with astreptavidin-phosphatase conjugate. Both the Coomasie-stained gel andthe blot demonstrated that VibB was successfully purified with biotinaffinity chromatography. In addition to high affinity methods, nativeproteins were isolated using non-denaturing purification, for instance,the affinity between carbohydrate-tagged proteins (i.e. β-mannosylatedproteins) and lectin linked-agarose resins (i.e., concanavalin A). Boundprotein was eluted off the agarose with a gradient of carbohydrate(i.e., mannose for beta-mannosylated proteins), and the purified proteinwas identified with SDS-PAGE and blot against a lectin peroxidaseconjugate (i.e., concanavalin A-peroxidase conjugate). This protocolproduced pure, non-denatured VibB tagged with mannose. This protocol canbe conducted with any affinity tag and molecular binding partner,including mannose-concanavalin A, peptide-antibody, and orpeptide-protein interactions. These results were reproduced usingmannose-linked CoA tagging to VibB with Sfp, isolating on ConcanavalinA-linked agarose column, and eluting with increasing concentrations offree mannose. This technique has the benefit of providing non-denaturedprotein, which can be further manipulated by enzyme activity assays toreporter individual domains, modules, or full synthase activity.

A 200 μL aliquot of cell culture induced with IPTG to overexpressrecombinant EntB or VibB was combined with 40 μL of biotinylated-analogof CoA and 14 of 1 mg/mL purified Sfp and allowed to react for 30 min atroom temp in the dark. 20 μL agarose-immobilized Streptavidin (4 mg/mLStreptavidin on 4% beaded agarose) was added to each sample andincubated at 4° C. for 1 hour with constant vigorous shaking. Aftercentrifugation at 14,000×g for 1 min, the supernatant was decanted andthe samples were washed 3 times with a solution containing 100 mMTris-Cl pH 8.4 and 1% SDS in water. After washing, the samples wereboiled in 50 mL SDS sample buffer for 10 min, centrifuged, and thesupernatant run on a 12% Tris-Glycine gel.

Example 12 Removal of Tag

Once proteins containing carrier proteins have been isolated, removal ofthe tagged 4′-phosphopantetheine-labeled moiety can be performed inorder for the carrier proteins to resume natural activity. This can beaccomplished with a phosphodiesterase that cleaves the phosphate linkagebetween the serine of the carrier protein and the tagged pantetheine. Inparticular, acyl-carrier-protein phosphodiesterase (ACP-PDE), used innatural systems to remove 4′-phosphopantetheine from fatty acid acylcarrier proteins, can be used for this purpose.

Example 13 Kinetic Analysis

Proteins identified, cloned and/or isolated through this study can alsobe used to determine kinetic properties of a given synthetic system.Herein, the loading and transfer properties of identified and purifiedFA, PK, and NRP synthases can be determined in vitro. Such studies canbe used to quantify the efficiency of a given PPTase/carrier proteinpair as well as to determine the efficiency of PPTase activity withindividual domains, individual modules, multiple modules, or completebiosynthetic systems. PPTase activity can be simply assayed through thefluorescent labeling technique described herein. Time course experimentscan be conducted to determine kinetic measurements of K_(out) and K_(m)values for individual carrier protein substrates or for individualfluorescent CoA derivatives. These techniques can also be used todetermine kinetic constants for inhibitors of the4′-phosphopantetheinylation process. These studies would involve timecourse experiments followed by protein precipitation via trichloroaceticacid or ammonium sulfate, wash, and fluorescent intensity measurement oftagged proteins. In addition, equilibrium based techniques such asequilibrium dialysis can also be used to identify the amount of reporteruptake as given by concentration of crypto-synthase. These data canyield rate information for further studies.

Example 14 Mechanistic Studies

Three major activities can be simply analyzed through biochemicaltechniques: these include (but are not limited to) posttranslationalmodification, amino acid or acyl monomer loading, condensation orketosynthase, and thioesterase activity. For instance, a module isolatedfrom a transgenic expression system and purified using mannosylatedtagging, concanavalin A-agarose affinity, and untagged using a PDEasecan be subsequently analyzed for in vitro 4′-phosphopantetheinylationkinetic rates with a PPTase and a fluorescent CoA derivative with a timecourse study. Subsequently the crypto-synthase (prepared by incubatedwith CoA and a PPTase) can be reported for loading in vitro: adenylation(in NRP synthase systems) or acyltransferase (in PK and FA synthasesystems) activity. Here, the isolated crypto-enzymes are incubated withradiolabeled amino acids and ATP (in NRP synthase systems) orradiolabeled malonyl CoA or methylmalonyl CoA (in PK and FA synthases).These experiments can be analyzed by SDS-PAGE and phosphorimaging todetermine whether the carrier protein domain is properly loaded with theproper monomer. This experiment can also be carried out with othertechniques, for instance using radiolabeled pyrophosphate with NRPsynthases and isolating ATP to probe for pyrophosphate exchange. Shouldenzymes be properly loaded, condensation activity (for NRP systems) orketosynthase (for PK and FA systems) can be studied next. Usingradiolabeled monomers pre-loaded onto the carrier proteins, acondensation/ketosynthase reactions can be identified between modules byTCA precipitation and SDS-PAGE and phosphorimaging. Alternatively,N-acetylcystamine thioesters of monomers or oligomers can be used toprobe internal condensation or ketosynthase activities in a synthase.Thioesterase activities are frequently reported with the use ofN-acetylcystamine thioesters of linear precursors and analyzed forcyclization or hydrolysis activity with chromatographic and massspectroscopy methods.

Example 15 Serially Addressable Fusion Protein-Tag (SAFP-TAG) FusionProteins

The methods of the present invention can be used to construct theSerially Addressable Fusion Protein-Tag (SAFP-TAG) fusion proteinsystem. A fusion protein system was created for these studies. One ofthe smallest polyketide carrier proteins, frnN, the frenolicin acyl CPfrom S. roseofulvus, contains 83 amino acids and demonstrates robustexpression in E. coli from a C-terminal histidine-tagged expressionvector called XA. A construct XA-frnN was modified at the 3′-end of thegene to convert it to a C-terminal fusion vector pDESTc-frnN. To createthe N-terminal fusion, the gene was subcloned to include the naturalstop codon back into XA and modify the construct at the 5′-end of thegene to create pDESTn-frnN. These two destination vectors were then usedto create a variety of fusion proteins from both eukaryotic andprokaryotic genes.

Modifying enzymes have been screened for optimal labeling kinetics. Over200 PPTase sequences have been annotated in the Genbank, and thousandsmore are accessible from NRP and PK expressing organisms. 15-20 of thesePPTases were cloned and sequenced from several bacterial and filamentousfungal species. Literature precedent has demonstrated that some PPTasesdisplay selective recognition of CP domains. For example, while it iswell established that the E. coli PPTase EntD, responsible for modifyingEntB, it is not sufficient for other secondary metabolic CP domains.This mechanism allows the selectivity to be engineered with termsregulating the choice of CP domain and reporter undergoing the labelingreaction.

Organisms with PPTase sequences in Genbank were obtained from theAmerican Type Culture Collection (ATCC), grown with appropriateconditions, and genomic DNA was isolated through a general benzylchloride procedure followed by amplification, cloning, and expression.These studies will be followed by PPTase activity studies involvingfluorescent and chemical reporters of various sizes and chemicalattributes.

Affinity reporters can be screened for manipulation of tagged fusionproteins. Several fluorescent and affinity reporter molecules have beenused. However, almost any biocompatible molecule can be attached to theCP domain in the compositions and methods of the present invention. Avariety of CoA reporter analogs will be synthesized for visualizationand affinity uses. These will include, but are not limited to, peptidetags, such as poly-histidine; carbohydrate tags, such as cellulose andsialyl-Lewis^(x); metal-tags, such as chelated mercury and nickel; DNAtags containing both single- and double-stranded fusions; lipid tags,including myristate, palmitate, and other bioactive fatty acids;radioactive tags with ³H, ³⁵S, ³²P, or ¹⁴C labeled molecules.

Example 16 Synthesis and Evaluation of Bioorthogonal Pantetheine Analogsfor in Vivo Protein Modification

In vivo carrier protein tagging has recently become an attractive targetfor the site-specific modification of fusion systems and new approachesto natural product proteomics. A detailed study of pantetheine analogswas performed in order to identify suitable partners for covalentprotein labeling inside living cells. A rapid synthesis ofpantethenamide analogs was developed and used to produce a panel whichwas evaluated for in vitro and in vivo protein labeling. Kineticcomparisons allowed the construction of a structure-activityrelationship to pinpoint the linker, dye, and bioorthogonal reporter ofchoice for carrier protein labeling. Finally bioorthogonal pantetheineanalogs were shown to target carrier protein with high specificity invivo, and undergo chemoselective ligation to reporters in crude celllysate. The methods demonstrated here allow carrier proteins to bevisualized and isolated for the first time without the need for antibodytechniques and set the stage for the routine use of carrier proteinfusions in chemical biology.

Recent years have seen intense research effort focused towardsdevelopment of new methods for the study and manipulation of covalentlymodified proteins, with particular attention given to in vivomethodologies. Bertozzi et al., Nat. Chem. Biol. 1:13, 2005. Fluorescentprotein fusions and antibody conjugates provide powerful tools forprotein imaging and manipulation. Tsien, Annu. Rev. Biochem. 67:509,1998; Lippincott-Schwartz et al., Science 300:87, 2003; Fritze et al.,Meth. Enzymol. 327:3, 2000 and Massoud et al., Genes Dev. 17:545, 2003.However drawbacks of these methods, such as structural perturbationssometimes induced by large fusions and general membrane impermeabilityof antibodies, have lead researchers to devise methods for thesite-specific modification of proteins by small-molecule probes. Ideallythese probes should be low molecular weight, covalent in nature, andpossessed of fluorescence or affinity properties allowing for facileimaging and manipulation. One such technique was recently introduced todemonstrate cellular uptake and covalent modification of carrier proteinfusions by pantetheine analogs. Clarke et al., J. Am. Chem. Soc.127:11234; 2005. These coenzyme A (CoA) precursors were shown topenetrate the cell membrane and be transformed into fully formed CoAderivatives via the endogenous CoA metabolic pathway, whereupon theywere transferred to a carrier protein by the promiscuousphosphopantetheinyltransferase (PPTase) Sfp. This advance allows carrierprotein labeling, a technique first developed from cell lysates andsince demonstrated on the cell surface, to be performed within the cell,opening the door for more sophisticated labeling systems. La Clair etal., Chem. Biol. 11:195, 2004; Yin et al., J. Am. Chem. Soc. 126:7754,2004; Yin et al., J. Am. Chem. Soc. 126:3570, 2004; George et al., J.Am. Chem. Soc. 126:8896, 2004; Yin et al., Chem. Biol. 12:199, 2005 andVivero-Pol et al., J. Am. Chem. Soc. 127:12770, 2005. Recentdevelopments have seen the trimming of the carrier protein domain downto just eleven amino acids, offering a fusion tag of the size andflexibility to be competitive with contemporary tagging systems andfurther highlighting the importance of techniques for the labeling ofintracellular carrier proteins. Yin et al., Proc. Natl. Acad. Sci.U.S.A. 102:15815, 2005.

Example 17 Strategies for Site-Specific Labeling of Proteins In Vivo

Several strategies for site-specific labeling of proteins in vivo havebeen previously demonstrated. Examples include Bertozzi's manipulationof the sialic acid biosynthetic pathway for the introduction of keto andazido functionalized cell-surface glycoproteins, Cravatt's introductionof azido/alkyne functionalities by covalent irreversible inhibition ofprotein active sites, and Hsieh-Wilson's chemoenzymatic introduction ofa keto-functionality for capture of O-GlcNAc-modified proteins. Mahal etal., Science 276:1125, 1997; Yarema et al., J. Biol. Chem. 273:31168,1998; Saxon and Bertozi, Science 287:2007, 2000; Speers and Cravatt,Chem. Biol. 11:535, 2004; Speers et al., J. Am. Chem. Soc. 125:4686,2003; Alexander and Cravatt, Chem. Biol. 12:1179, 2005; Hwan-Ching etal., J. Am. Chem. Soc. 126:10500, 2004; Khidekel et al., Proc. Natl.Acad. Sci. U.S.A. 36:13132, 2004. In each of these examples the proteinis not directly labeled with a fluorescence or affinity tag, but rathera unique and biologically inert chemical functionality is introduced.This functionality can then undergo reaction with exogenously deliveredreporters to label the protein of interest for detection and/orisolation, depending on the nature of the reporter. Advantages of thistwo-step labeling process include (i) better uptake of smaller probesdue to increased membrane permeability, (ii) increased incorporation ofprobes into native biosynthetic pathways due to greater similarity tonatural substrate, and (iii) the ability to conjugate a protein tovirtually any reporter possessing reactivity with the bioorthogonalfunctionality. Bertozzi et al., Nat. Chem. Biol. 1:13, 2005; Speers andCravatt, Chem. Biol. 11:535, 2004; Speers et al., J. Am. Chem. Soc.125:4686, 2003; Alexander and Cravatt, Chem. Biol. 12:1179, 2005. Here afull study of simplified pantetheine analogs that harness the power ofsuch bioorthogonal ligation reactions is presented. First the synthesisof simplified pantetheine analogs via a one-step reaction withpantolactone was optimized. Next, the specificity of the CoAbiosynthetic pathway is probed by a small panel of these simplifiedsubstrates. Finally, the utility of this strategy was validated bydemonstrating and comparing the delivery of bioorthogonal chemicalfunctionalities to carrier proteins in vitro and in vivo and using thenewly tagged carrier proteins for two widely applied chemoselectiveligations: the reaction of ketones and hydroxylamines to form oximes,and the Cu(I)-catalyzed azide-alkyne [3+2] cycloaddition reaction(“click” chemistry). This new ability to manipulate bioorthogonallytagged carrier protein in vivo promises to be a valuable tool for bothnew approaches to natural product proteomics as well as the study ofnovel intracellular carrier protein fusion systems.

Example 18 Analog Synthesis Pantolactone-Ring Opening

In efforts to address CoA biosynthesis with novel analogs, the synthesisof pantetheine and phosphopantetheine analogs that could be assembled ina manner analogous to peptide library synthesis was initiallyinvestigated. Mandel et al., Org. Lett. 6:4801, 2004. This necessitatedaddressing the synthetic challenges associated with pantolactone, namelythe lability of the α-proton following protection of the pantolactonesecondary alcohol. At this point, the presumption was made that theidentity of cystamine and β-alanine were necessary for turnover by theCoA metabolic pathway. However in vitro studies and recent work by Lee,indicated that little selectivity is gained through specificinteractions between the β-alanine moiety of pantothenate and PanK, thefirst enzyme in the CoA biosynthetic pathway and the gatekeeper fordownstream metabolism. Virga et al., Bioorg. Med. Chem. 14:1007, 2006;Jackowski and Rock, J. Bacteriol. 148:926, 1981. Further, E. coli PanK(CoAA) was found to catalyze the phosphorylation of pantetheine andanalogs with variations at the cystamine moiety almost as well aspantothenate itself. Worthington and Burkart, Org. Biomol. Chem. 4:44,2005. Given this newly revealed permissiveness in the CoA biosyntheticpathway, it was reasoned that simplified analogs of pantetheine could beused for both in vitro and in vivo applications. Elimination of theamide bond between cystamine and β-alanine significantly simplifiessynthetic access to reporter-modified pantetheine analogs by reducingoverall molecule polarity and solubility issues, eliminatingtime-consuming protection/deprotection steps of the 1,3-diol, andreplacing the multiple peptide coupling and purification steps ofprevious syntheses with a simple one-step nucleophilic ring-opening ofpantolactone. Clarke et al., J. Am. Chem. Soc. 127:11234, 2005. Withthis in mind the aletheine moiety (N-(β-Alanyl)-β-aminoethanethiol) wasmimicked with more synthetically flexible polyethylene glycol (PEG)linkers. In addition to the synthetic utility of this substitution, PEGspacers have the advantages of increasing aqueous solubility of smallmolecule probes and distancing reporter labels from labeled protein withthe effect of both enhancing secondary detection properties and reducingany negative effect of reporter/protein interactions. Kumar and Aldrich,Org. Lett. 5:613, 2003. These advantages were incorporated into thedesign of an ideal, synthetically straightforward, biodetectiblepantetheine analog.

In order to quickly access a large selection of analogs it was deemedappropriate to first revise the current methodology for pantothenamidesynthesis. Previous protocols calling for the base-promoted nucleophilicring opening of pantolactone by an amine could be subject toracemization or hampered by long reaction times (>24 hrs). Virga et al.,Bioorg. Med. Chem. 14:1007, 2006; Dueno et al., Tet. Lett. 40:1843,1999; Michelson, Biochim. Biophys. Acta, 93:71, 1964; Moffatt andKhorana, J. Am. Chem. Soc., 83:663, 1961. To address these problems,microwave-assisted organic synthesis was employed. By using(S)-(−)-α-methylbenzylamine one can test a variety of conditions fortheir ability to open pantolactone with a fairly hindered chiralnucleophile and analyze enantiopurity by ¹H-NMR (Table 1, see supportinginformation for ¹H-NMR data). Bertozzi et al., Nat. Chem. Biol. 1:13,2005.

TABLE 1 Data table for 1-step synthesis of pantetheine analogs vianucleophilic ring opening of pantolactone.

Amine Solvent Temp (° C.) Time (hr) Yield

EtOH 160 (a) 0.5 91%

DMF 165 (a) 0.5 63%

THF 110 (a) 0.5 44%

EtOH 160 (a) 0.5 42%

EtOH 160 (a) 0.5 82%

EtOH 160 (a) 0.5 75%

EtOH Reflux (b) 7 97%

Me0H Reflux (b) 7 84%

CH₃CN Reflux (b) 22 83%

DME Reflux (b) 12 30% (a) Microwave-assisted. (b) Thermal condition.

The study showed pantolactone to be surprisingly robust to a variety ofconditions, and reaction times could be reduced nearly 50-fold comparedto previous preparations with retention of optical purity. As expectedfrom the hypothesized transition state of this reaction, protic solventsproved ideal for nucleophilic ring-opening, with ethanol providing thebest balance of energy-absorbance and solubilization. Moving from thismodel-system to usefully functionalized amines, it was shown that alkyne(41), PEG (44), and fluorophore (20) containing pantetheine analogscould be synthesized in good to moderate yield within 30 minutes usingmicrowave-assistance. Interestingly a very recent report also presentedethanol as the solvent of choice for this transformation under thermalconditions; however without the addition of any base these large-scalesyntheses suffered from very long reaction times (72-120 hrs). Krause etal., Syn. Comm. 36:365, 2006. Accordingly the ideal microwave reactionconditions were also tested under simple reflux. Triethylamine proved tobe a sufficient base, as replacement with Hunig's base showed nosignificant effect on reaction outcome. Stronger bases were avoided.Again it was found that reflux of (S)-(−)-α-methylbenzylamine withexcess pantolactone and triethylamine provided pantetheine analogs withno apparent racemization in excellent yields in 7-12 hours. Thisalternative synthesis provides another avenue for analog preparation incases where the reporter or linker is sensitive to decomposition undermicrowave conditions. Microwave-assisted conjugation of7-dimethylaminocoumarin-4-acetic acid containing amines to pantolactoneresulted in the formation of unidentified decomposition products. Forthese couplings the classical condition (MeOH, NEt₃, reflux) was used.

Example 19 Synthesis of Bioorthogonal and Fluorescent PantetheineAnalogs

The general strategy for chemoenzymatic synthesis of CoA analogs isdepicted in FIGS. 7 and 8. FIG. 7 shows structures of pantetheineanalogs and biotin detection agents used in this study. FIG. 8 shows ageneral strategy for in vivo labeling of carrier protein by pantetheineanalogs. Virtually any mono-protected amine (21) can be transformed intoa pantetheine analog (25) by the three-stepcoupling/deprotection/ring-opening sequence. Cellular uptake andbiosynthetic processing by CoAA, CoAD, and CoAE yields the CoA analog28, which is then transferred to the carrier protein by a PPTase toyield bioorthogonally labeled carrier protein 29. After cell lysis thiscarrier protein can now be conjugated to the reporter of choice via anappropriate chemoselective ligation reaction.

First a mono-protected amine (depicted in this example by N-Bocethylenediamine 21) is conjugated to the biodetectible tag of choice bystandard peptide coupling conditions. After deprotection, this amine canbe conjugated to either pantolactone 24 through nucleophilic ringopening or pantothenic acid via EDAC mediated coupling. The newly formedpantetheine analog is then processed via stepwise conversion by CoAA,CoAD, and CoAE to form CoA analog 28, which is subsequently transferredto a conserved residue of the carrier protein by a PPTase to producereporter-modified crypto-carrier protein 30.

Analogs 12-18 were synthesized by this route in order to test thepermissibility of CoA biosynthesis toward unnatural pantetheine analogs,particularly the effect of changes in the β-alanine/cystamine region(Scheme 3). Three parallel amino-protecting group strategies (azide,Boc, Alloc) were chosen based on the commercial availability (32a),simple synthesis from literature preparations (33c, 34e), and orthogonalprotecting group traits (32b, 33d) of the specified diamines. Thisstrategy allowed compound 12 to be synthesized in two steps from N-Bocethylenediamine conjugated 7-dimethylaminocoumarin-4-acetic acid (DMACA)36c. La Clair et al. ChemBioChem 7:409, 2006. Acid-catalyzeddeprotection afforded the free amine, which performed nucleophilic ringopening of pantolactone to afford the final product in 85% yield.Compound 13 was synthesized as previously described, while dansylatedpantetheine 14 was synthesized by an analogous route. Clarke et al., J.Am. Chem. Soc. 127:11234, 2005. Compounds 15 and 16 were chosen to probethe effect of replacement of the strong H-bond accepting carbonyl andH-bond donating nitrogen of the natural substrate amide with weak H-bondaccepting ether oxygens. Their synthesis made use of a commonorthogonally protected diamine 48 (see supporting information), whichunderwent differential deprotection to give mono-protected diamines 32band 33d. Subsequent EDAC mediated conjugation to dye 31, azido/Bocdeprotection by standard conditions, and nucleophilic ring opening ofpantolactone afforded enzyme probes 15 and 16. Preliminary studies ofPEG-linked-pantoic acid conjugates showed good activity in in vitroassays, leading us to synthesize 17 in a 41% overall yield through ananalogous dye conjugation/deprotection/nucleophilic ring-openingsequence starting with the previously described N-Alloc diaminoethyleneglycol 34e. Compound 18 was chosen to test the limits of linker-lengthin CoA biosynthesis and was easily attainable from the commerciallyavailable mono-azido/mono-amino terminal nonaethylene glycol 32a througha similar series of reactions.

Compounds 1-8 sought to create a wide-spectrum of pantetheine analogsincorporating some of the most commonly used bioorthogonal tags such asketones, azides, and alkynes. Particular attention was paid to theazido-moiety, where analogs replacing the β-Ala (1), cystamine (2), andthio-acetal (5) regions of natural pantetheine with terminal azidemoieties were synthesized, in addition to experimentation with short (7)and long (8) PEG-linked azido-pantetheine analogs. Compound 1 wassynthesized in one step from the nucleophilic ring-opening ofpantolactone by 2-azidoethanamine (42). Compounds 2 and 3 (Scheme 2)were synthesized by standard peptide coupling ofp-methoxybenzylacetal-protected (PMP) pantothenate 39 and thecorresponding alkynyl and azido-amines, followed by acidic deprotectionof the 1,3-diol. Compounds 4 and 5 again utilized 39 as a startingmaterial, which was coupled to 2-azidoethanamine, deprotected to thecorresponding amine, and coupled to the correspondingN-hydroxysuccinimidyl keto/azido (43a,b) ester. PEG linked pantetheineconjugates 6 and 7 (Scheme 3) were constructed in a similar fashion bynucleophilic ring-opening of pantolactone under microwave-conditions byN-Alloc protected diaminoethylene glycol 44 to give a commonintermediate, followed by Pd(PPh₃)₄ mediated deprotection and couplingof the purified free amine to an keto/azido acid activated as thesuccinimidyl ester. Finally, analog 8 was synthesized in one stepthrough microwave-assisted nucleophilic ring-opening of pantolactone bymono-azido/mono-amino terminated nonaethylene glycol 32a.

Example 20 In Vitro Pathway Uptake Kinetics with CoAA

As mentioned earlier, phosphorylation of the primary hydroxyl group ofpantothenate by the first protein in the CoA biosynthesis pathway, PanK,is believed to be the rate limiting step in vivo. Jackowski and Rock, J.Bacteria 148:926, 1981. Due to its role as the gatekeeper of CoAbiosynthesis, each of the new analogs was assayed for kinetic activitywith PanK (Table 2).

TABLE 2 Kinetic parameters of E. coli PanK with natural substrates andpantetheine analogs. Compound # k_(cat) (min⁻¹) K_(m) (μM) k_(cat)/K_(m)(s⁻¹ mM⁻¹) Pantothenate 31.27 ± 0.58 28.56 ± 1.77 18.25 ± 0.68 Pantetheine¹⁴ 19.2 ± 0.1  91 ± 10 3.53 ± 0.44 1 22.33 ± 1.54  692.3 ±89.13 0.54 ± 0.07 2 31.45 ± 0.75 32.85 ± 2.54 15.96 ± 0.76  3 28.017 ±0.42  43.53 ± 1.99 10.73 ± 0.32  4 20.64 ± 0.45 62.65 ± 3.89 5.49 ± 0.245 40.91 ± 1.12 71.89 ± 6.98 9.48 ± 0.52 6  1.60 ± 0.06 0.1955 ± 0.09 136.4 ± 10.63 7  6.16 ± 0.44  53.76 ± 11.16 1.91 ± 0.27 8 NA NA NA 1211.56 ± 0.57 37.24 ± 5.82 5.17 ± 0.51 13 19.16 ± 1.41 28.40 ± 6.92 11.25± 1.65  14 17.26 ± 0.62 27.33 ± 3.28 10.53 ± 0.76  15  1.36 ± 0.05  0.76± 0.32 0.03 ± 0.04 16  1.00 ± 0.03  0.14 ± 0.18 0.12 ± 0.01 17 10.06 ±0.43 51.18 ± 6.42 3.28 ± 0.28 18 NA NA NA

The assay was performed as previously described using the prototypicalbacterial PanK, CoAA from E. coli. Kumar and Aldrich, Org. Lett. 5:613,2003. The reaction of CoAA with the natural substrate pantothenate gavevalues that conformed to those previously reported in the literature.Worthington and Burkart, Org. Biomol. Chem. 4:44, 2005; Strauss andBegley, J. Biol. Chem. 277:48205, 2002. Pantothenate mimics 2 and 3 showk_(cat) and K_(m) values closely approaching those of the naturalsubstrate. As seen in previous studies pantetheine is also a substratefor CoAA, and compounds 4, 5, 13, and 14 show turnover near equal (4,13, 14) or exceeding (5) that of the natural substrate in these cases.Compounds 5, 13, and 14 are processed particularly efficiently by PanK.Conversely, compounds with PEG linkers between the pantoic acid moietyand the bioorthogonal terminus were poor substrates for CoAA. The lengthof the linker region was a strong factor in determining substratesuitability, with the longest compounds 8 and 18 proving such poorsubstrates that kinetic data could not be generated. Shorter PEG linkedcompounds (7 and 17) were viable substrates in the assay, but turnedover at a rate 2-5 fold less than derivatives containing β-Ala/diaminelinkers. PEG linked pantoic acid conjugate 6 differs only from 7 by theexchange of an azide for an acyl substitutent, but shows markedlydecreased kinetic activity.

To isolate and investigate the effect of H-bond accepting heteroatoms inthe linker region, pantetheine analogs 15 and 16 were synthesized. Thesecompounds were designed to replace the amide bond between β-Ala andcystamine of pantetheine with a single ether oxygen, allowinginvestigation of subtle substrate-enzyme interactions in the PanK activesite. Virga et al., Bioorg. Med. Chem. 14:1007, 2006. Surprisingly,these compounds were extremely poor substrates. While compounds with theaforementioned short PEG linkers (7, 17) showed K_(m) values two-foldhigher than natural substrate pantothenate, and compounds withtraditional β-Ala/diamine linkers were either the same as (13, 14) ortwo fold higher (4, 5), the K_(m) values for 15 and 16 were 100-foldlower. Turnover for these compounds was proportionately low, indicativeof tight binding. To test if these compounds were acting as inhibitorsof CoAA, a competitive kinetic assay was set up using pantotenate as thesubstrate. The results (see supporting information) show that 15 acts asa non-competitive inhibitor, suggesting that it may bind the allostericregulation site of CoAA. Ivey et al., J. Biol. Chem. 279:35622, 2004.Investigation of these compounds as potential inhibitor scaffolds isongoing. Azide 1, which omitted entirely the β-Ala/carbon diamine or PEGlinkages of other analogs showed turnover within the range of thenatural substrate; however K_(m) was 20-fold higher than pantothenatesuggesting deletion of an interaction involved in active site binding.Another interesting pantoic acid analog 12, which shortened the pantoicacid-reporter linker length to four carbons and reversed the carbonyland amide β-Ala/cystamine linkage of natural pantetheine, showed lowerturnover and catalytic efficiency than analogs containing the naturalβ-Ala/pantoic acid linkage (5, 13, 14).

In Vitro Pathway Uptake: Gel Shift The conversion of apo-ACP to holo-ACPor reporter-modified crypto-ACP causes a change in the mobility of theprotein on a non-denaturing polyacrylamide gel. Virga et al., Bioorg.Med. Chem. 14:1007, 2006. In order to assay each compound for activitythroughout the entire co-opted CoA pathway covalently modified carrierprotein was run on a native PAGE gel and compared mobility to apo-ACP(FIG. 9 a). Pantetheine analogs were reacted as previously reported withthe enzymes CoAA-E to create CoA analogs, followed by the addition ofthe PPTase Sfp and apo-ACP. Clarke et al., J. Am. Chem. Soc. 127:11234,2005. As seen in FIG. 9 a, all of the compounds tested demonstrated somechange in mobility with relation to apo-ACP. For most compounds fullconversion to crypto-ACP is obtained; however analogs with long PEGlinkers (8, 18) give multiple bands on the gel that suggest apo-proteinremains.

To confirm the results from this assay, the reaction mixtures weresubjected to matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS). The MALDI-MS data (FIG. 10) confirms that allpantetheine analogs in the panel are indeed converted into CoAderivatives and transferred onto carrier protein in vitro. Apo-ACP (4a)shows a characteristic peak with a mass of 8505 Da. Compound 13 wasreacted with the CoAA biosynthesis enzymes, ACP, and Sfp as describedabove and the reaction mix analyzed by MALDI without furtherpurification. As seen in FIG. 10 b shows the reaction with the CoAanalog of 13 causes the expected mass change of 604 mass units. PEGanalogs 17 and 18 (FIGS. 10 c,d) also show the expected mass shifts of559 units and 916 units respectively, corresponding to the formation ofcrypto-carrier protein. Only in the case of the PEG-linked derivatives 8and 18 does the conversion appear significantly incomplete, supportingthe results of the gel shift assay.

In Vitro Pathway Uptake: Biodetectability Having confirmed that thepantetheine analogs were suitable substrates for the CoA biosynthesisenzymes and PPTase/carrier protein reaction, the biodetectability ofeach analog was investigated. The fluorescent analogs (12-18) weredetected by UV visualization on PAGE gels as previously described.Clarke et al., J. Am. Chem. Soc. 127:11234, 2005. Bioorthogonally taggedpantetheine analogs 1-8 were detected by chemoselective ligation to theappropriate alkoxyamine/azide/alkyne functionalized biotin followed byPAGE and visualization by western blotting and incubation withstreptavidin-conjugated alkaline phosphatase. Keto-pantetheine compounds4 and 6 were reacted overnight with biotin hydroxylamine (9) at roomtemperature, while pantetheine analogs with azide and alkynefunctionalities were reacted with the corresponding alkynyl/azido(10/11) biotin following the procedure of Alexander. Alexander andCravatt, Chem. Biol. 12:1179, 2005. Inspection of the fluorescent gels(FIG. 9 b) and western blots (FIG. 9 d) confirmed the results of the gelshift assay and mass spec data, indicating biodetectible covalentmodification of carrier protein in vitro for compounds 1-8 and 12-18.

FIG. 9 shows in vivo and in vitro activity of pantetheine analog panel.(a) Analogs were assayed for gel shift after reaction with CoAbiosynthesis enzymes (CoAA/D/E), PPTase (Sfp) and carrier protein (E.coli ACP). Conversion of apo-ACP to reporter modified crypto-ACP causesa change in the mobility of the protein on native PAGE. (b) In vitromodification of the carrier protein VibB by reaction with fluorescentpantetheine analogs, CoA biosynthesis enzymes (CoAA/D/E) and Sfp. (c) Invivo modification of carrier protein by incubation of fluorescentpantetheine analogs with E. coli overexpressing VibB and the PPTase Sfp.(d) In vitro modification of VibB by reaction with bioorthogonalpantetheine analogs, CoA biosynthesis enzymes (CoAA/D/E) and Sfp.Labeled carrier protein is visualized by chemoselective ligation to theappropriate biotin reporter (9-11) followed by SDS-PAGE, blotting ontonitrocellulose, and incubation with streptavidin-linked alkalinephosphatase. (e) In vivo modification of carrier protein by incubationof bioorthogonal pantetheine analogs with E. coli overexpressing VibBand the PPTase Sfp. Visualization as in (d).

FIG. 10 shows in vitro labeling of ACP. Apo-ACP (a) is reacted withpantetheine analogs, CoA biosynthetic enzymes (CoAA/D/E) and the PPTaseSfp. Fluorescent compounds 13 (b), 17 (c), and 18 (d) are all shown tobe converted to CoA analogs and modify ACP by gel and mass spectralanalysis.

Example 21 In Vivo Uptake

The library of pantetheine analogs was tested for integration into theE. coli CoA pathway using an in vivo assay. Clarke et al., J. Am. Chem.Soc. 127:11234, 2005. E. coli overexpressing the carrier protein VibBand the PPTase Sfp were incubated with 1 mM of each compound in 1 ml ofculture. After 4 hours of growth cells were pelleted, washed, and lysed.Lysate from these cultures was run on SDS-PAGE gels and detectioncarried out as described for the in vitro studies. Compounds 2, 3, 5,12, and 13 demonstrated detectable modification of VibB in vivo (FIG. 9c/e). Keto-pantetheine analog 4 was also detectable, but showed muchweaker labeling than the similarly linked azido-analog 5 (FIG. 9 e). Thecompounds most active in vivo show a strong correlation with CoAAkinetic profile. To verify the results of the gels and blots, samples ofcrude lysate were assayed by MALDI-MS (FIG. 11). For MALDI-MS analysisdoubly transformed E. coli containing plasmids for the carrier proteinFren and the PPTase Sfp were used. As seen in FIG. 11 a compound 13 wastaken up by the cell, processed into a CoA analog and attached ontoFren. The apo peak can be seen at 8570 mass units. A small amount ofholo-carrier protein is also visible at 8910 Da. This peak arises fromthe fact that natural pantetheine is available in the cell and readilyligated by PPTases to the overexpressed apo-Fren. Carrier proteinmodified with 13 can be seen at 9179 Da giving the expected 609 massunit change. Similarly mass spec analysis of cell lysate afterincubation of bioorthogonal pantetheine analog 2 with Fren/Sfpoverexpressing E. coli shows an observable 315 Da shift of the known apopeak indicating formation of alkyne-modified crypto-carrier protein.Subjection of the same crude cell-lysate to click reaction conditionswith biotin reporter 11 resulted in another 317 Da mass shift,indicating successful formation of biotinylated Fren via aCu(I)-catalyzed [3+2] cycloaddition process.

FIG. 11 shows in vivo carrier protein labeling. The carrier protein Frenis labeled in vivo by incubation of E. coli overexpressing Fren and Sfpwith (a) a fluorescent pantothenate analog 13 and (b) a bioorthogonallytagged analog 2. Click reaction of alkyne-modified crypto-carrierprotein with biotin reporter 11 affords triazole-linked biotinylatedcarrier protein (c), resulting in the expected shift in mass andallowing protein visualization by western blot.

Example 22 Insights into CoAA Substrate Specificity from Kinetic, InVivo, and In Vitro Analyses

Comparisons of the kinetic, in vitro, and in vivo assay results fordifferent members of the panel yields important insight into thestructure-activity relationships between E. coli PanK and pantetheineanalogs. Despite the poor performance of compounds 1, 6-8, 12, and 15-18in the PanK kinetic assay, all were shown to be converted to CoA analogsand loaded onto the carrier protein ACP by Sfp in vitro. This is both atestament to the incredible efficiency of Sfp in transferring unnaturalCoA derivatives to apo-carrier proteins and a further demonstration ofthe utility of the chemoenzymatic approach to synthesis of unnatural CoAanalogs. As mentioned above this finding greatly simplifies thesynthetic task of constructing CoA derivatives for in vitro applicationswhich utilize carrier protein tagging. George et al., J. Am. Chem. Soc.126:8896, 2004; Yin et al., Chem. Biol. 12:199, 2005 and Vivero-Pol etal., J. Am. Chem. Soc. 127:12770, 2005. This allows access to virtuallyany reporter labeled-CoA analog from a mono-protected amine in threesteps, one of which can be expedited using microwave technology.Additionally the subtle substrate preferences Sfp has been shown toexhibit in systems incorporating multiple carrier proteins can be usedfor selective coding based on differential phosphopantetheinylation byunnatural CoA analogs, adding another layer of complexity to in vitroand cell-surface carrier protein fusion systems. Mercer et al.,ChemBioChem., 8:1335, 2005.

Detailed analyses of the kinetic data demonstrate several importantrelationships. In general compounds containing a similar β-Ala linkerregion to natural pantethenate show the best kinetic profiles. In allcompounds of the panel the pantoic acid region (C1-N5, numbering fromthe terminal primary hydroxyl of pantetheine) was conserved. Lee et al.indicated low binding of PanK inhibitors which lacked a proton-donatingamide NH at the N8 position, pointing to a model of thePanK-ADP-pantothenate ternary complex in which the C-7 acid ofpantothenate acts as an H-bond donor towards two key residues. Virga etal., Bioorg. Med. Chem. 14:1007, 2006; Ivey et al., J. Biol. Chem.279:35622, 2004. The results verify the importance of this interaction.Compounds 1 and 3 contain the same 2-azidoethanamine-derived terminalazide, differing only in the β-Ala linkage of 3. This change results ina 10-fold increase in K_(m) and near 20-fold increase in catalyticefficiency, indicating much better binding of the substrate with theH-bond donating β-Ala linker. Pantetheine analogs containing ethyleneglycol based linkers incapable of acting as efficient H-bond donors(6-8, 17-18) showed similarly poor kinetics compared with thosecontaining β-Ala linkers. Compounds 15 and 16 provide perhaps thestrongest evidence of the importance of this interaction, losing almostall substrate activity when reducing the strength of the electron pairdonor and removing the H-bond donating NH completely from thepantetheine analog. Interestingly these compounds show markedlydifferent kinetics than PEG-linked pantetheine 17, which differs by onlytwo atoms, demonstrating the limitations of this model in predictingeffects caused by alternate variables such as the reduced rotationaround an sp² hybridized carbon at C8 and addition of an extra H-bondaccepting heteroatom further down the linker. The kinetics of analog 12,in which the connectivity of the amide at C8 and N9 of the naturalsubstrate pantetheine is reversed, indicate that there is someflexibility in the pocket around this position. Compound 12 shows goodbinding but slow turnover, with a catalytic efficiency poorer than anyof the compounds with the H-bond donator in its natural position (2-5,13-14) but better than every compound in which the H-bond donating NH isabsent (1, 6-8, 15-18). While the general trend toward an H-bond donoreffect is large, other interactions resulting from the proximity of thearomatic coumarin-reporter molecule to the active site in this analogmust also be considered.

Several other trends which may be important for future design of carrierprotein tags are of interest. In general, analogs terminating in alkynesshow better kinetic parameters than azides, and azides better thanketones. For PEG-based pantetheine derivatives, chain length proved animportant factor, with longer chains showing negligible activity withCoAA and poor in vivo protein tagging. Appending a different dye to theend of the pantetheine had no statistically significant impact on CoAAkinetics; however substitution of DMACA with dansyl lead to a completeloss of in vivo activity, suggesting a possible lack of a viablemembrane-transport mechanism for dansyl-pantetheine analogs or anintracellular degradation process. It has been shown previously thatDMACA is an excellent dye for in vivo applications. La Clair et al.,ChemBioChem 7:409, 2006. The kinetics of β-Ala containing bioorthogonalpantetheines (2-5) compared with β-Ala containing pantetheines in whicha fluorescent reporter was directly appended (13-14) showed slightlybetter turnover and similar catalytic efficiency. On the whole thebinding site of PanK beyond the β-Ala moiety appears to be quitepromiscuous, with little kinetic effect observed on substitution of thehexanediamine linker of 13 with a ethylenediamine linker or substitutionof the ethylenediamine linker of 14 with a short (8-atom) PEG linker.This comparison refers to kinetic data compiled for alternatively linkeddansyl and DMACA-pantetheine analogs in reference 14.

Perhaps the most important conclusion that can be drawn from the assayresults is that the in vivo activity of a pantetheine analog has adirect correlation with kinetic activity with PanK. Analogs 2, 3, 4, 5,12, and 13 were shown to be biodetectible in E. coli by Western blotanalysis and fluorescence visualization. These show a k_(cat) between11.5-40.9 min⁻¹ and k_(cat)/K_(m) of 5.2-16.0 compared with the valuesof 31.5 min⁻¹ (k_(cat)) and 3.5 s⁻¹ mM⁻¹ (k_(cat)/K_(m)) for naturalpantetheine. In order for a pantetheine analog to be processed into aCoA analog in vivo it must have comparable or better kinetics with CoAAthan natural substrate pantetheine of the host. Given that enterococciproduce far more pantetheine than they require for primary metabolism,any modified pantetheine analog must make extremely efficient use ofCoAA in order for CoA conversion and subsequent protein labeling tooccur at detectable levels. Rock et al., J. Biol. Chem. 275:1377, 2000.E. coli has been shown to have a pantetheine import system, the panFsymporter, which also may exert some selectivity in the import ofanalogs and thus influence the ability of pantetheine analogs to beintegrated into the CoA pathway in vivo. Jackowski and Alix, J.Bacteriol. 172:3842, 1990. However previous studies showed that whileoverexpression of the panF gene resulted in elevated pantothenateuptake, a concurrent increase in CoA production was not observed,indicating PanK activity as the principal regulator of CoA biosynthesis.With that in mind these kinetic parameters should prove useful for thefuture design and assay of in vivo carrier protein tags.

Example 23 In Vitro Applications and In Vivo Cell-Surface Labeling ofCarrier Protein Fusions

The molecules described here have varied applications and provideinsight valuable in the expanding field of proteomics. For in vitroapplications and in vivo cell-surface labeling of carrier proteinfusions, all pantetheine analogs studied herein are efficientlyconverted into CoA analogs and tethered to the protein via the four-stepenzymatic pathway. This alternative methodology negates the purificationsteps necessary in production of maleimide-CoA analogs from commercialsources, allows almost any variance of the chemical identity of thelinker region, and provides an economical substitute for producing largeamounts of CoA analogs in cases where large quantities of the desiredCoA-maleimide-reporter conjugate may be prohibitively expensive. Inaddition the expansion of analogs with bioorthogonal reporters allowsfor increased detection and sensitivity. However despite these subtleadvances, it is in the prospect of in vivo labeling that these toolsbecome particularly important. The covalent modifications describedherein have practical value in the study of in vivo activity of proteinsand a place among the ever increasing myriad of proteomic techniquesused to study them.

Of particular importance are the chemoselective ligation reactionsdemonstrated by the ketone, azide, and alkyne protein labels. Thesetools allow carrier proteins to be visualized and isolated for the firsttime without the expense and complication of antibody techniques. Whilethe survey of bioorthogonal coupling partners was not exhaustive, thefunctionalities introduced should be applicable to other publishedmethods. For instance, one can easily envisage analogs 3 and 5 beingmodified by Bertozzi's covalent Staudinger ligation with areporter-conjugated triarylphosphine analog for in vivo applications inwhich more stringent Cu(I)-catalyzed click chemistry conditions are notideal. Kohn and Breinbauer, Angew. Chem. Int. Ed. Engl. 43:3106, 2004.The PEG-incorporating pantetheine analogs proved non-amenable to in vivoprotein labeling, most likely to deletion of a crucial H-bondinginteraction. Yet while PEG analogs are often useful for distancingreporter labels from the protein of interest for downstreammodification, most likely they are not a necessity in this instance byvirtue of the 4′-phosphopantetheine moiety. Indeed the4′phosphopantetheine is commonly believed to be appended to carrierproteins as a means to distance substrates and products from the proteincore, a concept reinforced by recent structural studies of the fattyacid synthase. Simon Jenni et al., Science 311:1258, 2006; Maier et al.,Science 311:1263, 2006.

A detailed investigation of pantetheine analogs has been performed toidentify suitable partners for covalent protein labeling inside livingcells. A rapid synthesis of pantethenamide analogs was developed forthis purpose and used to produce a panel which was evaluated for invitro and in vivo protein labeling. Kinetic comparisons allowed theconstruction of a structure-activity relationships to pinpoint thelinker, dye, and bioorthogonal reporter of choice for protein labeling.Finally bioorthogonal pantetheine analogs were shown to target carrierproteins with high specificity in vivo and undergo chemoselectiveligation to reporters in crude cell lysate. The principal barrier to theutilization of small-molecule probes in cellular contexts is a shortageof site-specific protein labeling methodologies. Chen and Ting, Curr.Opin. Biotech. 16:35, 2005. The detailed understanding of the kineticparameters and structural limitations of pantetheine analogs sets thestage for the routine use of 4′-phosphopantetheine analog labeling inchemical biology.

Abbreviations: CoA, Coenzyme A; PPTase, phosphopantetheinyltransferase;O-GlcNAc, O-linked β-N-acetylglucosamine; β-Ala, β-alanine; PanK,pantothenate kinase; PEG, polyethylene glycol; Pantolactone,(D)-(−)-pantolactone; Boc, tert-butyl carbamate; Alloc, allyl-carbamate;DMACA, 7-dimethylaminocoumarin-4-acetic acid; CP, carrier protein; ACP,fatty acid synthase acyl carrier protein from E. coli; VibB,vibriobactin synthase carrier protein from Vibrio cholerae; Fren,frenolicin synthase carrier protein from Streptomyces roseofulvus.

General procedures for microwave assisted ring-opening of pantolactone,experimental details for the synthesis of compounds 1-18, kinetic, invitro, and in vivo assay details, full author listings for references 19and 21 and ¹³C-NMR spectra of all final compounds and syntheticintermediates is available free of charge via the Internet athttp://pubs.acs.org.

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety for all purposes asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference for allpurposes.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method to generate analogs of coenzyme A in a cell, comprising:reacting pantetheine or a derivative thereof with a reporter to formlabeled pantetheine or a derivative thereof; contacting the cell withthe labeled pantetheine or derivative thereof; phosphorylating thelabeled pantetheine or derivative thereof to form labeledphosphopantetheine; adenylating the labeled phosphopantetheine orderivative thereof to form a labeled-dephosphoCoenzyme A or a derivativethereof, and; phosphorylating the 3′-hydroxyl of thelabeled-dephosphoCoenzyme A or derivative thereof to form alabeled-coenzyme A analog or a derivative thereof.
 2. The method ofclaim 1, wherein the pantetheine or derivative thereof comprises threemodules.
 3. The method of claim 2, wherein the three modules areselected from a ω-functionalized amine, a sidechain, an α-aminoacidβ-aminoacid, a linker, or a modulator.
 4. The method of claim 3, whereinthe ω-functionalized amine, the sidechain, the α-aminoacid β-aminoacid,the linker, or the modulator comprise a reporter (R).
 5. The method ofclaim 3, wherein the modulator is a dihydroxy acid, an aminohydroxyacid, a pantoic acid, or a homolog or derivative of pantoic acid.
 6. Themethod of claim 1, wherein the reporter is attached to the labeledcoenzyme A analog by an azide-alkyne cycloaddition reaction.
 7. Themethod of claim 1, wherein the reporter is attached to the labeledcoenzyme A analog by a ketone-hydroxylamine reaction.
 8. The method ofclaim 1, wherein the reporter is an affinity reporter, a coloredreporter, a fluorescent reporter, a magnetic reporter, a radioisotopicreporter, a peptide reporter, a metal reporter, a nucleic acid reporter,a lipid reporter, a glycosylation reporter, a reactive a reporter, anenzyme inhibitor, or a biomolecular substrate.
 9. The method of claim 1,wherein the cell is a prokaryotic cell.
 10. The method of claim 1,wherein the cell is a eukaryotic cell.
 11. The method of claim 1,further comprising reacting the labeled-coenzyme analog or derivativethereof with a carrier protein domain to form a labeled protein.
 12. Themethod of claim 11, wherein the carrier protein domain comprises afusion construct between a peptide or a carrier protein domain and aprotein of interest.
 13. The method of claim 11, wherein the reaction iscatalyzed by a phosphotransferase.
 14. The method of claim 13, whereinthe phosphotransferase is 4′-phosphopantetheinyltransferase.
 15. Themethod of claim 1, wherein the labeled-phosphopantetheine analog can beused to identify proteins, characterize proteins, inhibit proteins,activate proteins, or examine the structure of proteins.
 16. The methodof claim 1, wherein the labeled-dephosphocoenzyme A analog can be usedto identify proteins, characterize proteins, inhibit proteins, activateproteins, or examine the structure of proteins.
 17. The method of claim1, wherein the labeled-coenzyme A analog and related isomers of thelabeled-coenzyme A analog can be used to identify proteins, characterizeproteins, inhibit proteins, activate proteins, or examine the structureof proteins.